CN111655843A - NK or T cells and uses thereof - Google Patents

NK or T cells and uses thereof Download PDF

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CN111655843A
CN111655843A CN201880083143.XA CN201880083143A CN111655843A CN 111655843 A CN111655843 A CN 111655843A CN 201880083143 A CN201880083143 A CN 201880083143A CN 111655843 A CN111655843 A CN 111655843A
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cell
cells
cancer
carcinoma
mice
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A·曼托瓦尼
M·摩尔格拉
C·噶兰达
E·伯纳维他
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Thumanitas, University of
Humanitas Mirasole SpA
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Abstract

The present invention relates to an isolated stable or transient Il-1R8 deficient human cell which is a Natural Killer (NK) cell or a T cell, or a medical use thereof, preferably for the treatment of tumors and infections.

Description

NK or T cells and uses thereof
Technical Field
The present invention relates to an isolated stable or transient Il-1R8 deficient human cell which is a Natural Killer (NK) cell or a T cell, or a medical use thereof, preferably for the treatment of tumors and infections.
Prior Art
Interleukin-1receptor 8(IL-1R8, also known as the monoimmunoglobulin IL-1R-related receptor, SIGIRR, or TIR8[ NCBI gene ID: 59307; NM-001135053.1 → NP-001128525.1; NM-001135054.1 → NP-001128526.1; NM-021805.2 → NP-068577.2, the sequence is shown below:
NCBI reference sequence NP-001128525.1
GenPept-like protein mapping
Single Ig IL-1-associated receptor for NP-001128525.1 [ Homo sapiens ]
MPGVCDRAPDFLSPSEDQVLRPALGSSVALNCTAWVVSGPHCSLPSVQWLKDGLPLGIGGHYSLHEYSWV
KANLSEVLVSSVLGVNVTSTEVYGAFTCSIQNISFSSFTLQRAGPTSHVAAVLASLLVLLALLLAALLYV
KCRLNVLLWYQDAYGEVEINDGKLYDAYVSYSDCPEDRKFVNFILKPQLERRRGYKLFLDDRDLLPRAEP
SADLLVNLSRCRRLIVVLSDAFLSRAWCSHSFREGLCRLLELTRRPIFITFEGQRRDPAHPALRLLRQHR
HLVTLLLWRPGSVTPSSDFWKEVQLALPRKVQYRPVEGDPQTQLQDDKDPMLILRGRVPEGRALDSEVDP
DPEGDLGVRGPVFGEPSAPPHTSGVSLGESRSSEVDVSDLGSRNYSARTDFYCLVSKDDM
(SEQ ID NO:29)
NCBI reference sequence NP-001128526.1
GenPept-like protein mapping
Single Ig IL-1-associated receptor for NP-001128526.1 [ Homo sapiens ]
MPGVCDRAPDFLSPSEDQVLRPALGSSVALNCTAWVVSGPHCSLPSVQWLKDGLPLGIGGHYSLHEYSWV
KANLSEVLVSSVLGVNVTSTEVYGAFTCSIQNISFSSFTLQRAGPTSHVAAVLASLLVLLALLLAALLYV
KCRLNVLLWYQDAYGEVEINDGKLYDAYVSYSDCPEDRKFVNFILKPQLERRRGYKLFLDDRDLLPRAEP
SADLLVNLSRCRRLIVVLSDAFLSRAWCSHSFREGLCRLLELTRRPIFITFEGQRRDPAHPALRLLRQHR
HLVTLLLWRPGSVTPSSDFWKEVQLALPRKVQYRPVEGDPQTQLQDDKDPMLILRGRVPEGRALDSEVDP
DPEGDLGVRGPVFGEPSAPPHTSGVSLGESRSSEVDVSDLGSRNYSARTDFYCLVSKDDM
(SEQ ID NO:30)
NCBI reference sequence NP-068577.2
GenPept-like protein mapping
Single Ig IL-1-associated receptor for NP-068577.2 [ Homo sapiens ]
MPGVCDRAPDFLSPSEDQVLRPALGSSVALNCTAWVVSGPHCSLPSVQWLKDGLPLGIGGHYSLHEYSWV
KANLSEVLVSSVLGVNVTSTEVYGAFTCSIQNISFSSFTLQRAGPTSHVAAVLASLLVLLALLLAALLYV
KCRLNVLLWYQDAYGEVEINDGKLYDAYVSYSDCPEDRKFVNFILKPQLERRRGYKLFLDDRDLLPRAEP
SADLLVNLSRCRRLIVVLSDAFLSRAWCSHSFREGLCRLLELTRRPIFITFEGQRRDPAHPALRLLRQHR
HLVTLLLWRPGSVTPSSDFWKEVQLALPRKVQYRPVEGDPQTQLQDDKDPMLILRGRVPEGRALDSEVDP
DPEGDLGVRGPVFGEPSAPPHTSGVSLGESRSSEVDVSDLGSRNYSARTDFYCLVSKDDM
(SEQ ID NO:31)
]) Are members of the IL-1receptor (ILR) family, which have distinct structural and functional characteristics, and act as down-regulators of ILR and Toll-like receptor (TLR) downstream signaling pathways and inflammation1。
The IL-1 system has a central role in both the natural and acquired immune responses, and it is tightly controlled at different levels both extracellularly and intracellularly by antagonists, decoy receptors, scavengers, dominant negative molecules, mirnas, and other mechanisms.
IL-1R8/TIR8/SIGIRR are atypical receptors, a novel down-regulator of the ligand-mediated inflammatory and adaptive responses of the IL-1 system.
The IL-1R8/TIR8/SIGIRR gene is located on human chromosome 11 and on murine chromosome 7, and the protein (410 amino acids) is constructed by a single Ig extracellular domain with several N-and O-glycosylation sites, a transmembrane domain, an intracellular conserved TIR domain and a 95 amino acid long C-terminal tail.
IL-1R8/TIR8/SIGIRR is widely expressed, especially in epidermal tissues such as kidney, digestive tract, liver and lung, and in lymphoid organs by lymphocytes.
IL-1R8/TIR8/SIGIRR has been reported to inhibit NF-kB, JNK and mTOR kinase activation following stimulation by IL-1 receptors or TLR family members. It negatively regulates signaling activated by IL-1R1, IL-18R, ST2, and several TLRs, such as TLR1/2, TLR3, TLR4, TLR7, and TLR9, which are members of the IL-1receptor family. Possible molecular mechanisms include: interference with IL-1R1 and IL-1RAcP dimerization by the extracellular Ig domain of IL-1R8/TIR8/SIGIRR, and binding of the TIR-containing aptamer molecule by the TIR domain, rendering it no longer capable of signaling.
Natural Killer (NK) cells are natural lymphocytes that mediate resistance to pathogens and contribute to the activation and orientation of adaptive immune responses2-4. NK cells mediate resistance to hematopoietic cell tumors, but are generally thought to be less effective in solid tumor carcinogenesis5-7。
Several lines of evidence suggest that IL-1R8 interferes with the binding of aptamer molecules containing TIR components to signaling receptor complexes of the ILR or TLR family, regulates downstream signaling, and thus has negative control over inflammatory and immune responses, as well as T helper cell polarization and function1,8。
CD4+ T lymphocytes have previously been shown to express IL-1R8(Garland C et al, Trends Immunol (2009); Gulen et al, Immunity (2010); Bulek et al, J Immunol (2009); Bozza et al, J Immunol (2008)). These studies report that IL-1R8 is a negative regulator of CD4+ T lymphocytes and that IL-1R8 has increased helper functions when genetically silenced in mice. Helper activity can be achieved by different T subsets, whereas in the T lymphocyte subset cytotoxicity is mostly achieved by the CD8+ T subset. The molecular mechanisms regulating the cytotoxic potential of CD8+ T lymphocytes differ from those involved in CD4+ T lymphocytes, as well as the functional activities of these two cell types, since CD4+ T cells have helper functions, whereas CD8+ T cells are cytotoxic. Therefore, IL-1R8 is in the fineIn addition, IL-1R8 is a co-receptor of IL-1R5/IL-18R α for IL-37, and is essential for the anti-inflammatory activity of this human cytokine9. Dysregulated activation of ILR or TLR ligands in IL-1R8 deficient mice with exacerbated inflammation and immunopathology, including selected cancer or autoimmune diseases10。
WO2005084696 describes the use of a medicament which interacts with TIR8/SIGIRR for the preparation of a therapeutic composition for the treatment of gastrointestinal inflammation and for stimulating mucosal or epidermal immunity.
WO2007034465 describes the new finding that IL-1F5(IL-1) and its derivative polypeptide bind to the receptor SIGIRR, and the binding interaction can modulate the immune response by stimulating the production of cytokine IL-4. This induces an anti-inflammatory immune response. PPAR γ has also been shown to be a key mediator of downstream signaling of SIGIRR following activation of IL-1F5 ligand. Modulation of the immune response occurs in neuronal tissue following binding of IL-1F5 to SIGIRR, and corresponding methods of treating neurodegenerative diseases are described.
There remains a need for methods of treating tumors with NK or T cells.
Summary of The Invention
The inventors found that IL-1R8 acts as a checkpoint for NK cell maturation and effector function. Its gene blockade releases NK-cell mediated resistance to liver carcinogenesis, hematopoietic liver and lung metastases, and cytomegalovirus infection.
Detailed Description
The inventors have discovered that IL-1R8 has NK cell antitumoral and antiviral activity checkpoint effects. The genetic inactivation of IL-1R 8in NK cells suggests possible translation in NK cell-based cell therapy.
The inventors herein describe:
IL-1R8(mRNA and protein) is expressed by human and murine NK cells, IL-1R8 expression being upregulated during NK cell maturation;
IL-1R8 deficiency in mice is associated with an increased frequency of mature NK subgroups in blood and lymphoid organs;
IL-1R 8-deficient NK cells produce elevated levels of IFN γ and exhibit increased cytotoxic activity when stimulated in vitro with suitable cytokines, including IL-18 (an IL-1family member that acts through IL-18R and is down-regulated by IL-1R 8);
in three different cancer models (3-MCA-induced sarcoma lung metastasis, colon cancer-derived liver metastasis and DEN-induced hepatocellular carcinoma), IL-1R 8-deficient mice were protected: the inventors observed a reduced incidence of primary tumors, a reduced volume or invasiveness for hepatocellular carcinoma, and a reduced number and volume of metastases in lung and liver metastasis models;
NK cell depletion abrogated the protective effects observed in IL-1R8 deficient mice.
The inventors herein also show that IL-1R8 deficient NK cells significantly and strongly reduced the number and volume of metastases in NK cell adaptive metastasis experiments in preclinical models of mouse lung and liver metastases (fig. 3 i-j). This suggests that IL-1R8 deficiency is associated with increased anti-tumor activity of NK cells.
In addition, they also found that the expression level of IL-1R8 was inversely proportional to NK cell activation in humans (fig. 21), and that IL-1R8 was associated with increased NK cell activation by siRNA genetic inactivation in human NK cells, as shown by IFN γ production (fig. 2m) and CD69 expression, indicating that IL-1R8 is a negative regulator of NK cell activation, and that its inactivation releases human NK cell effector function.
IL-1R8 was also expressed in CD8+ T cells, suggesting a broad utility of IL-1R8 as a checkpoint molecule in NK and T cells and possibly involving IL-1R8 inactivation (FIG. 1 a).
The inventors herein also show that IL-1R8 deficiency is associated with increased proliferation, maturation and functional activation of CD8+ T cells.
Accordingly, an object of the present invention is an isolated human cell which is a natural killer cell (NK) or T cell, wherein the cell has a stable or transient defect in IL-1R8 expression and/or activity. The T cell is preferably a CD8+ T cell.
The cells preferably produce a large number of effector molecules which are involved in the anti-tumor immunity, preferably interferon gamma (IFN γ) and/or telomerase B and/or FasL and/or express maturation markers, preferably CD44, at a higher level than cells expressing IL-1R 8.
Preferably, the above cells are also deficient in the expression and/or activity of at least one checkpoint of NK cell maturation and/or effector function. The at least one checkpoint of NK cell maturation and/or effector function is preferably selected from: CIS, KIRs, PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT.
Still another object of the present invention is a cell population comprising the above NK cells and/or T cells, a composition comprising the above defined cells or the above defined cell population, preferably further comprising at least one physiologically acceptable carrier.
The above defined cells or populations, or compositions are preferably for use as a medicament, more preferably for use in the treatment and/or prevention of tumours and/or metastases, or microbial or viral infections.
The above defined cells or populations or compositions are preferably used for Adoptive Cell Transfer (ACT), cell therapy, mismatched bone marrow transplantation, mismatched NK cell infusion or Cytokine Induced Killer (CIK) cell infusion. The NK or T cells are preferably previously isolated from the same treated individual or from a different individual.
Another object of the present invention is a repressor or inhibitor of IL-1R8 expression and/or activity for medical use, preferably for use in the treatment and/or prevention of tumors and/or metastases, or microbial or viral infections.
The repressor or inhibitor is preferably at least one molecule selected from the group consisting of:
a) an antibody or fragment thereof;
b) a polypeptide;
c) a small molecule;
d) a polynucleotide encoding the antibody or polypeptide or functional derivative thereof;
e) polynucleotides, such as antisense constructs, antisense oligonucleotides, RNA interference constructs or siRNA,
e) a vector comprising or expressing a polynucleotide as defined in d) or e);
f) CRISPR/Cas9 components, e.g., sgRNA
g) A host cell genetically engineered to express said polypeptide or antibody, or which comprises a polynucleotide as defined in d) or e) or a module in f).
Preferably the polynucleotide is an RNA inhibitor, preferably selected from: siRNA, miRNA, shRNA, stRNA, snRNA, and antisense nucleic acids, more preferably the polynucleotide is at least one siRNA selected from AGU UUC GCG AGC CGA GAU CUU (SEQ ID NO: 1); UAC CAG AGC AGC ACG UUG AUU (SEQ ID NO: 2); UGA CCC AGG AGU ACUCGU GUU (SEQ ID NO: 3); CUU CCC GUC GUU UAU CUC CUU (SEQ ID NO:4) (all 5.
The repressor or inhibitor is preferably used for NK and/or T cell and/or Adoptive Cell Transfer (ACT), cell therapy, mismatched bone marrow transplantation, mismatched NK cell infusion or cytokine-induced killer (CIK) cell infusion. Preferably the repressor or inhibitor is used in NK and/or T cell therapy. The host cell is preferably an NK or T cell.
Still another object of the invention is a pharmaceutical composition comprising a repressor or inhibitor as defined above, and at least one pharmaceutically acceptable carrier, optionally together with a therapeutic agent.
The above-mentioned tumor is preferably a solid tumor or a hematological tumor, preferably selected from the following: colon/rectal cancer, adrenal cancer, anal cancer, biliary tract cancer, bladder cancer, bone cancer, adult brain/CNS tumor, childhood brain/CNS tumor, breast cancer, male breast cancer, unknown primary cancer, castleman's disease, cervical cancer, endometrial cancer, esophageal cancer, ewing's tumor family, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, hodgkin's disease, kaposi's sarcoma, kidney cancer, larynx and hypopharyngeal cancer, leukemia, Acute Lymphocytic Leukemia (ALL), acute myelogenous leukemia (AML, including myelogenous sarcoma and skin leukemia), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous (CML) leukemia, chronic myelomonocytic leukemia (ml), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, lymphoma, skin lymphoma, cutaneous lymphoma, small cell lung cancer, colon cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, adenocystic carcinoma of the nasal cavity and sinuses, nasopharyngeal carcinoma, neuroblastoma, non-hodgkin's lymphoma in children, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma-adult soft tissue cancer, skin cancer-basal and squamous cells, skin cancer-melanoma, skin cancer-merkel cells, small bowel cancer, gastric cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, uveal melanoma, vaginal cancer, vulval cancer, waldenstrom's macroglobulinemia, wilms ' tumor, more preferably the tumor is a solid tumor, preferably colorectal cancer, and the metastasis is a lung or liver metastasis.
The infection is preferably caused by one of the following viruses or bacteria: herpes viruses, preferably cytomegalovirus, Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Hepatitis B Virus (HBV), West Nile Virus (WNV), Salmonella, Shigella, Legionella, mycobacteria.
Another object of the invention is a method for obtaining a cell or population, or composition, as defined above, comprising stably or transiently inhibiting the expression and/or function of IL-1R 8in NK or T cells or cell populations, and optionally expanding the silenced population in vitro. The T cell is preferably a CD8+ T cell. The method is preferably an in vitro or ex vivo method.
The NK or T cells or population are preferably previously purified from isolated Peripheral Blood Mononuclear Cells (PBMC), optionally expanded in vitro, preferably using recombinant human interleukin-2 (rhIL-2).
Preferably, the above method further comprises inhibiting or suppressing the expression and/or function of at least one other checkpoint of NK cell maturation and/or effector function. The at least one checkpoint of NK cell maturation and/or effector function is preferably selected from: CIS, KIRs, PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT.
In the above methods, the stable or transient inhibition or repression of IL-1R8 expression and/or function in NK or T cells is preferably achieved with at least one repressor or inhibitor as defined above.
In the present invention, "CD 8+ T cells" include cytotoxic T cells (also referred to as TCs, cytotoxic T lymphocytes, T-killer cells, cytolytic T cells, CD8+ T cells or killer T cells), T lymphocytes that kill cancer cells (a type of white blood cells), infected cells (particularly by viruses), or cells that are otherwise damaged. Most cytotoxic T cells express T Cell Receptors (TCRs) that recognize specific antigens. Intracellular antigens bind to MHC class I molecules, which bring the antigen to the cell surface and are therefore recognised by T cells. For the TCR to bind to MHC class I molecules, the former must be accompanied by a glycoprotein called CD8, which binds to a constant portion of the MHC class I molecule. Thus, these T cells are referred to as CD8+ T cells.
For the purposes of the present invention, cells deficient in IL-1R8 expression and/or activity are those in which IL-1R8 (protein and/or mRNA) levels are reduced or inhibited permanently or transiently in their entirety. Cells deficient in IL-1R8 expression and/or activity can be obtained by, for example, silencing with a CRISPR/Cas9 system, siRNA, peptides or antibodies that interfere with interaction with other ILR/TLR receptors. The deficient cell can be transformed with a sgRNA, preferably delivered into the cell with a CRISPR-Cas9 system.
In one embodiment, the NK and/or T cell deficient in IL-1R8 expression and/or activity does not express detectable IL-1R 8. In another embodiment, the NK and/or T cell deficient in IL-1R8 expression and/or activity does not express an immunologically detectable IL-1R 8. In one embodiment, the NK and/or T cell deficient in IL-1R8 expression and/or activity does not express detectable IL-1R8 mRNA. NK and/or T cells deficient in IL-1R8 expression and/or activity (or lacking functional IL-1R8) can be prepared by any conventional method. In some embodiments, cells deficient in IL-1R8 expression and/or activity are obtained by inhibiting or blocking IL-1R8 expression by, for example, gene deletion, gene disruption, siRNA, shRNA, or antisense approaches. In other embodiments, a cell deficient in IL-1R8 expression and/or activity is obtained by inhibiting or blocking IL-1R8 activity, e.g., by an IL-1R8 antagonist or antibody. In some embodiments, the cell deficient in IL-1R8 expression and/or activity is obtained by genetically modifying an immune cell to block expression of endogenous IL-1R 8. Although homologous recombination is used in some cases, non-homologous end joining is used in certain cases to edit the genome. Any suitable scheme for modifying the genome of a particular immune cell is useful, although in particular embodiments genetic modification is achieved by engineered nucleases, such as zinc finger nucleases (ZFPs), TALE-nucleases (TALENs), or CRISPR/Cas nucleases. Engineered nuclease technology is based on the engineering of naturally occurring DNA binding proteins. For example, engineering of homing endonucleases with modulated DNA binding specificity has been described (see Chames, et al (2005) Nucleic Acids Res.33(20): el 78; Arnould, et al (2006) J.mol.biol.355: 443-. In addition, engineering of ZFPs has been described. See, e.g., US 6,534,261; US 6,607,882; US 6,824,978; US 6,979,539; US 6,933,113; US 7,163,824; and US 7,013,219.
All the above definitions of "cells deficient in expression and/or activity" also refer to (optionally modified) cells deficient in "at least one checkpoint expression and/or activity of NK cell maturation and/or effector function".
The term "checkpoint of NK cell maturation and/or effector function" includes fundamental molecules of immune-mediated response regulation, such as the following molecules: CIS (cytokine inducible SH 2-containing protein), KIRs (killer immunoglobulin-like receptors), PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT (Hsu J et al, JCI (2018) https:// doi.org/10.1172/JCI99317; Guillerey C et al, Nat Immunol (2016) https:// doi.org/10.1038/ni.3518; Delconte RB et al, Nat Immunol (2016) https:// doi.org/10.1038/ni.3470). PD-1 blockade is known to be beneficial for immune reactivation and therefore protective and curative in tumor models and tumor patients; other molecules (i.e., CTLA-4, PD-L1, KIRs, TIM-3, NKG2A, CD96, TIGIT, CIS) regulate different pathways and act by different mechanisms and were also previously considered inhibitory molecules in NK cells. Most of them have been used clinically, others are under development (e.g., CIS and CD 96). PD-1 is the most clinically used checkpoint molecule for which there is a tool for preclinical studies in mice. The role of PD-1 as an NK cell checkpoint has recently been published (Hsu J et al, JCI (2018)). PD-1 is expressed in terminally differentiated and depleted cytotoxic lymphocytes, induced by chronic activation, and acts as an immunosuppressive mechanism in the tumor microenvironment (Freeman GJ et al, JEM (2000)). PD-1-dependent immunosuppressive activity is determined by the ligand (PD-L1) expressed on target cells, particularly tumor cells (Freeman GJ et al, JEM (2000); Hsu J et al, JCI (2018)). Thus, inhibition of the PD-1/PD-L1 axis with checkpoint inhibitors (anti-PD-1 or anti-PD-L1 blocking antibodies) can only be achieved in the presence of cytotoxic cell types (e.g., NK cells, CD8+ T cells) and targets (e.g., tumor cells).
For the purposes of the present invention, an "effector molecule involved in antitumor immunity" is a molecule that mediates the underlying mechanism of an immune response against tumor cells. Preferably it is interferon gamma (IFN-. gamma.), telomerase B, FasL.
The cell population of the invention preferably comprises at least 50% NK cells and/or T cells as defined above.
In one embodiment, the composition or cell population as defined above comprises at least 50% or more NK and/or T cells deficient in IL-1R8 expression and/or activity. In another embodiment, the composition or population of cells comprises at least 70% or more NK and/or T cells deficient in IL-1R8 expression and/or activity. In another embodiment, the composition or population of cells comprises at least 80% or more IL-1R 8-expressing and/or activity-deficient NK and/or T cells.
The T cell of the invention is preferably CD8+T cells.
The cytokines were observed in vivo. Thus the expression "production of said cells" includes not only direct production but also indirect production of cytokines, involving the final effect of the tumor progression, with different control between the two animal groups.
All known methods for obtaining/expanding mature NK or T cells can be used. Several strategies have been developed for obtaining/expanding mature NK cells in vitro (see, e.g., Fang F. et al, Semin Immunol 31(2017) 37-54; Davis Z.B. et al, Semin Immunol 31(2017) 64-75).
For example, NK cells can be purified from PBMC and expanded in vitro with rhIL-2. Next, IL-1R8 can be silenced using any silencing method, such as the CRISPR/Cas9 system or siRNA, or neutralized with mAb. Preferably, cytokine pretreatment is contemplated, and NK or T cells may be injected into a patient by any convenient route of administration, for example, by intravenous or intra-arterial infusion. (see, e.g., Koehl U, et al, Front Oncol.2013, 5/17.d.; 3:118.doi:10.3389/fonc.2013.00118. Electroselection 2013.Granzim N. et al, Front Immunol.2017, 4/26.4/2017; 8:458.doi:10.3389/fimmu.2017.00458. Electroselection 2017).
In the context of the present invention, "IL-1R 8 activity" or "activity of IL-1R 8" includes, for example, interaction with other IL-1R family members and TLR family members, negative regulation of TLR family member activation and signaling, inhibition of NF-kB, JNK and/or mTOR kinase activation, negative regulation of IL-1receptor family members, for example IL-1R1, IL-18R, ST2, and TLRs, for example TLR1/2, TLR3, TLR4, TLR7 and/or TLR9 activated signaling.
IL-1R8 is a membrane receptor that interacts with other IL-1R family members and TLR family members, negatively regulating its activation and signal transduction. For example, the inventors inhibited IL-1R8 activity with gene silencing by genetic defects in mice and in humans using Dharmacon TMACCELL (TM) siRNA technology.
In addition, IL-1R8 activity can be inhibited or silenced using CRISPR/Cas9 systems, other siRNAs, peptides or antibodies that interfere with interaction with other ILR/TLR receptors, as described, for example, in Semin Immunol 31(2017), Fang F.et al, 37-54. In the present invention, the terms "active" and "functional" are interchangeable.
The NK cells of the invention include NK progenitor cells and mature functional NK cells.
NK progenitor cells can differentiate into mature and functional NK cells, recognizing the desired target (e.g., NKG2D, DNAM-1, NCRs, KIR-receptor) through surface-specific receptors known to experts in the field. These mature and functional NK cells can be produced in vitro by extending the culture period for 2-3 weeks. For cell therapy, however, injection of primitive progenitor cells and maturation in vivo is preferred. These NK cells are useful for the treatment of tumors, cancers, in particular leukemias, ovarian, colon and skin cancers, breast, brain and lung cancers, cervical cancers and metastases of all cancers, in particular liver metastases, and all viral diseases, in particular HCV, HIV and other chronic viral diseases.
The dosage of these viral compositions is generallyIndicates the number of viable cells present in the composition. The amount should be 1-9x106NK-initiating cells or>l-10x 108Mature NK cells or 1-9x106T cells/kg body weight of the individual to be treated.
Following pretreatment with cytokines, the NK cells of the invention may be infused into the patient by intravenous or arterial injection (see, e.g., Koehl U, et al, Front oncol.2013, 5 months and 17 days; 3:118.doi:10.3389/fonc.2013.00118.ecol 2013.Granzim n. et al, Front immunol.2017, 4 months and 26 days; 8:458.doi:10.3389/fimmu.2017.00458.ecol 2017).
Polynucleotides such as the siRNAs described above may further include dTdT or UU-3' overhangs, and/or nucleotide and/or polynucleotide backbone modifications as described elsewhere herein. For the purposes of the present invention, the term "polynucleotide" includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA, siRNA, shRNA) and DNA or RNA analogs generated using nucleotide analogs. The polynucleotide may be single-stranded or double-stranded. The RNAi inhibitor defined above is preferably capable of hybridizing to all or part of a particular target sequence. Thus, the RNAi inhibitor may be fully or partially complementary to all or part of the target sequence. RNAi inhibitors can hybridize to a particular target sequence under moderate to high stringency conditions. RNAi inhibitors can be determined by the specific sequence identity of the reverse complement of the sequence to which they are targeted. Antisense sequences typically have at least about 75%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% sequence identity to the reverse complement of their target sequence.
The terms polynucleotide and polypeptide also include derivatives and functional fragments thereof. Polynucleotides (e.g., inosine or phosphorothioate nucleotides) can be synthesized using oligonucleotide analogs or derivatives.
For the purposes of the present invention, the gene defined above (e.g., IL-1R8) is preferably identified as the sequence indicated by its NCBI gene ID and GenBank accession number. However, they also include the corresponding orthologous or homologous genes, isoforms, variants, allelic variants, functional derivatives, functional fragments thereof.
For the purposes of the present invention, the term "gene" also includes the corresponding orthologous or homologous genes, isoforms, variants, allelic variants, functional derivatives, functional fragments thereof. The expression "protein" is intended to also include the corresponding proteins encoded by the corresponding orthologous or homologous genes, functional mutants, functional derivatives, functional fragments or analogs, isoforms thereof.
In the present invention, the term "polypeptide" or "protein" includes:
i. intact proteins, allelic variants and orthologs thereof;
any synthetic, recombinant or proteolytic functional fragment;
any functional equivalent, such as a synthetic or recombinant functional analogue.
The term "analogue" as used herein refers to a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues, and/or wherein one or more amino acid residues have been deleted from the peptide and/or one or more amino acid residues have been added to the peptide. Such amino acid residue additions or deletions may occur at the N-terminus of the peptide and/or the C-terminus of the peptide.
A "derivative" may be a nucleic acid molecule, e.g.a DNA molecule, a nucleic acid molecule encoding or comprising the above-mentioned polynucleotide, or a polynucleotide of complementary sequence. In the present invention, the term "derivative" also refers to longer or shorter polynucleotides and/or polypeptides having a percent identity of, for example, at least 41%, 50%, 60%, 65%, 70% or 75%, more preferably at least 85%, such as at least 90%, even more preferably at least 95% or 100% to the sequences mentioned herein or to their complementary sequences, or to their DNA or RNA counterparts. The term "derivative" and the term "polynucleotide" also include modified synthetic oligonucleotides. The modified synthetic oligonucleotides are preferably LNA (locked nucleic acid), phosphorothioate or methylated oligomers, morpholino, 2' -O-methyl, 2' -O-methoxyethyl oligonucleotides and cholesterol-coupled 2' -O-methyl modified oligonucleotides (antagomers). The term "derivative" may also include nucleotide analogs, i.e., naturally occurring ribonucleotides or deoxyribonucleotides substituted with non-naturally occurring nucleotides. The term "derivative" also includes nucleic acids or polypeptides, equivalents or precursor sequences thereof, which can be generated by mutation of one or more nucleotides or amino acids in the sequence. The term "derivative" also includes at least one functional fragment of a polynucleotide. In the present invention, "functional" means, for example, "retaining its activity". The above antibodies include human and animal monoclonal antibodies or fragments thereof, single chain antibodies and fragments thereof and miniantibodies, bispecific antibodies, diabodies, triabodies, or dimeric, oligomeric or polymeric versions thereof. Also included are peptidomimetics or peptides derived from the antibodies of the invention, e.g., which include one or more CDR regions, preferably the CDR3 region. Also included are human monoclonal antibodies and peptide sequences, based on structural active linkages, generated by a manual modeling process (greenj. et al, j.med. chem.,1994, volume 37, page 1035-.
Preferably the antibody is selected from the group consisting of: intact immunoglobulin (or antibody), Fv, scFv (single chain Fv fragment), Fab, F (ab)'2An antibody-like domain, an antibody-mimicking domain, a single antibody domain, a multimeric antibody, a peptide or a proteolytic fragment comprising an epitope-binding region. The term "antibody" in the present invention is used in the broadest sense and includes a variety of antibodies and antibody mimetic structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, deimmunized antibodies, chimeric antibodies, nanobodies, antibody derivatives, antibody fragments, anticalin, designed ankyrin repeats (DARPins), affibodies, affilins, adhesins (affimers), affitines, alpha bodies (alphabodies), avimers, fynomers, minibodies (minibodies), and other binding domains, provided that they exhibit desirable binding activity to an antigen. An "antibody fragment" refers to a molecule other than an intact antibody, which molecule comprises a portion of an intact antibody that binds to an antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')2(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and antibody fragments. F of VH and VLv is also referred to as a "nanobody". The term "mimobody" refers to an organic compound or binding domain that is not a derivative of an antibody but specifically binds to an antigen in the same manner as an antibody. They include antiporters, designed ankyrin repeats, affibodies, affilins, avidin, affitine, alpha bodies, avimers, fynomers, minibodies, and the like. The term "chimeric" antibody refers to an antibody in which the heavy and/or light chain portions are derived from one particular source or species, while the remaining heavy and/or light chains are derived from a different source or species.
The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as described herein. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody-encoding sequence. The definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. In humans, the antibody isotypes are IgA, IgD, IgE, IgG and IgM. An antibody is "humanized" to refer to a chimeric antibody comprising the amino acid residues of the non-human hypervariable region (HVR) and the amino acid sequence from the remaining human region (FR: framework region). In certain embodiments, a humanized antibody can comprise substantially at least one entire variable region, typically two, in which all or substantially all of the HVRs (e.g., CDRs) correspond to a relative portion of a non-human antibody and all or substantially all of the FRs correspond to a relative portion of a human antibody. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. "humanized forms" of antibodies, e.g., non-human antibodies, refer to antibodies that have been humanized. "deimmunized" antibodies are antibodies that reduce immunogenicity by disrupting HLA binding, the fundamental requirement for T cell stimulation. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA-based methods, phage display methods, and methods using transgenic animals containing all or part of a human immunoglobulin locus. For the present invention, the antibody of the present invention includes the antibody modification according to the present invention which can maintain the above-mentioned specificity thereof. Such changes include, for example, conjugation to effector molecules such as chemotherapeutic or cytotoxic agents and/or detectable reporter moieties.
Bispecific antibodies are macromolecular heterobifunctional cross-linkers that have two different binding specificities within one molecule. Within this group, for example, bispecific (bs) IgG, bs IgM-IgA, bs IgA-dimer, bs (Fab')2,bs(scFv)2Diabodies and bs-bis-Fab Fc (Cao y. and Suresh m.r., Bioconjugate chem.,1998, vol 9, p 635-644).
Peptide mimetics refers to low molecular weight peptide components, which means structures that mimic the native peptide component, or a template that induces the formation of specific structures in adjacent peptide sequences (Kemp DS, Trends biotechnol.,1990, page 249-255). The peptide mimetic can be, for example, derived from the CDR3 domain. The methodological mutation analysis of a given peptide sequence, i.e. by alanine or glutamate scanning mutation analysis, can be used. Another possibility to improve the activity of certain peptide sequences is the use of peptide libraries in conjunction with high throughput screening.
The term antibody may also include substances obtained by analysis of data relating to structure-activity relationships. These compounds are also useful as peptidomimetics (Grassy G. et al, Nature Biotechnol.,1998, Vol.16, p. 748-752; Greer J. et al, J. Med. chem.,1994, Vol.37, p. 1035-1054).
The term antibody may also include proteins produced by expression of altered immunoglobulin-encoding regions in a host cell, e.g., "technically modified antibodies" such as synthetic antibodies, chimeric or humanized antibodies or mixtures thereof, or antibody fragments which partially or completely lack constant regions, e.g., Fv, Fab' or F (ab)2, and the like. In these techniques modified antibodies, for example, one or more portions of the light and/or heavy chains may be substituted. These molecules may, for example, comprise antibodies consisting of a humanized heavy chain and an unmodified light chain (or chimeric light chain), or vice versa. The term Fv, Fc, Fd, Fab, Fab 'or F (ab)'2As described in the prior art (Harlow E. and Lane D, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
The invention also encompasses the use of Fab fragments or F (ab)'2Fragments derived from a monoclonal antibody (mAb) directed against IL-1R8 or other NK cell maturation and/or effector function checkpoints. Preferably, the hetero-framework and constant regions are selected from the group consisting of human immunoglobulin classes and isotypes, such as IgG (subtypes 1-4), IgM, IgA and IgE. During the course of an immune response, a type switch of immunoglobulins may occur, for example a switch from IgM to IgG; where the constant region is exchanged from, for example, μ to y. Type conversion may also occur in a targeted manner by genetic engineering methods ("targeted type conversion recombination"), as is known in the art (Esser c. and radbreak a., annu. rev. immunol.,1990, volume 8, page 717-. However, the antibodies of the invention need not exclusively include human sequences of immunoglobulins.
Antibodies of the invention also include those antibodies whose binding characteristics have been improved by direct mutation, affinity maturation, phage display. Affinity or specificity may be altered or improved by mutations in the CDRs of any of the antibodies of the invention. The term "variable region" or "variable domain" refers to the heavy or light chain domain of an antibody that is involved in binding of the antibody to an antigen. The variable domains (or regions) of the heavy and light chains (VH and VL, respectively) of natural antibodies typically have similar structures, each domain comprising 4 framework conserved regions (FRs) and three hypervariable regions (HVRs see, e.g., Kindt et al, "Kuby Immunology", 6 th edition, w.h.freeman and co., page 91, 2007). Single VHOr VLThe domain may be sufficient to confer antigen binding specificity. In addition, libraries of complementary VL or VH domains can be screened by isolating antibodies that bind to a particular antigen using VH or VL domains from antibodies that bind to the antigen (see, e.g., Portolano et al, J.Immunol.150:880-887, 1993; Clarkson et al, Nature 352:624-628, 1991).
Antibody-like domains include binding proteins that are structurally related to antibodies, such as T cell receptors. The antibodies of the invention also include functional equivalents, which include polypeptides having amino acid sequences substantially identical to the amino acid sequences of the variable or hypervariable regions of the antibodies of the invention. "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the alignment of the sequences and introduction ofAfter gaps, if necessary, to achieve the maximum percent sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, and does not take into account any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve full-length maximum alignment of the compared sequences. The antibody of the present invention may have, for example, the following dissociation constant (K)D)：<100nM,<10nM,<1nM,<0.1nM,<0.01nM, or<0.001nM or less, e.g. from 10-8M to 10-13M, e.g. from 10-9M to 10-13And M. Recombinant and/or biotechnological derivatives as well as fragments of the above mentioned antibodies are also encompassed by the present invention, provided that the binding activity of the antibody and its functional specificity are maintained.
In the context of the present invention, "cancer" or "tumor" includes primary and metastatic tumors, as well as refractory tumors, solid or non-solid tumors. A further aspect of the invention is a nucleic acid encoding or hybridizing to the above-mentioned antibody or consisting of a corresponding degenerate sequence.
The scope of the present invention includes expression vectors encoding the above-described antibodies, preferably comprising the above-described nucleic acids. Also within the scope of the invention are host cells comprising the above-described nucleic acids or the above-described vectors.
The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells," which include transformed primary cells and progeny derived therefrom, not counting the number of steps. The nucleic acid content of the progeny is not necessarily identical to that of the parent cell and may comprise mutations. In the present invention, mutant progeny are included which have the same function or biological activity as screened or selected in the originally transformed cell. The nucleic acids of the invention may be used to transform a suitable mammalian host cell. Mammalian cells useful as expression hosts are well known and include, for example, CHO and BHK cells. Prokaryotic hosts include, for example, E.coli, Pseudomonas, Bacillus, and the like. The antibodies of the invention may be fused to additional amino acid residues, such as a tag to facilitate isolation. The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors in the form of self-replicating nucleic acid structures, as well as vectors that enter the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. These vectors are currently referred to as "expression vectors". Any suitable expression vector may be used, for example, a prokaryotic cloning vector such as a plasmid for E.coli, e.g., colE1, pCR1, pBR322, pMB9, pUC. Expression vectors suitable for expression in mammalian cells include derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences. The expression vector used in the present invention comprises at least one expression control sequence operably linked to a DNA sequence or fragment which must be expressed. Another object of the present invention is a pharmaceutical composition comprising at least the above-mentioned antibody or a synthetic or recombinant fragment thereof, and a pharmaceutically acceptable excipient, preferably said composition is for parenteral administration, preferably intravenous use. The compositions comprise an effective amount of an antibody and/or recombinant or synthetic antigen-binding fragments thereof. Pharmaceutical compositions are conventional in the art and can be prepared by those skilled in the art based on conventional general knowledge. Formulations for use in the treatments described herein may, for example, comprise the above-described antibody at a concentration of from about 0.1mg/ml to about 100mg/ml, preferably from 0.1 to 10mg/ml, more preferably from 0.1 to 5 mg/ml. In other formulations, the antibody concentration may be lower, for example at least 100 pg/ml. The antibodies of the invention are administered to a patient in one or more treatments. Depending on the type and severity of the disease, a dosage of about 1-20mg/kg of antibody may be administered, e.g., one or more administrations or continuous infusion. The antibodies of the invention may be administered in combination with other therapeutic agents, particularly with antibodies that neutralize other receptors involved in tumor growth or angiogenesis. Any method of administration may be used for administering the antibody of the present invention, and in particular, for example, administration may be oral, intravenous, intraperitoneal, subcutaneous, or intramuscular. The antibodies of the invention may be administered as conjugates that specifically bind to the receptor and release the toxic substance. In particular embodiments, the pharmaceutical compositions of the present invention may be administered in a single dosage form (e.g., tablets, capsules, pills, etc.). For pharmaceutical use, the compositions may be in the form of solutions, e.g., injectable solutions, emulsions, suspensions, and the like. The carrier may be any pharmaceutically suitable carrier. Preferably, the carrier used enhances the entry of the molecule into the target cell. In the pharmaceutical compositions of the invention, the inhibitor or repressor may be combined with other therapeutic agents, such as antagonists of other growth factor receptors involved in tumorigenesis or angiogenesis, e.g., VEGFR-2, EGFR, PDGFR, receptor kinase inhibitors, BRAF inhibitors, MEK inhibitors, immunomodulatory antibodies, anti-cancer drugs, such as: bevacizumab, ramucizumab, afidopril, sunitinib, pazopanib, sorafenib, cabozantinib, axitinib, regorafenib, nintedanib, lenvatinib, vemurafenib, dabrafenib, trametinib, chemotherapeutic agents, such as methylating agents (temozolomide, dacarbazine), platinum compounds (cisplatin, carboplatin, sariplatin), taxanes (taxane (paclitaxel), albumin bound taxanes, docetaxel), fluorinated pyrimidines (5-fluorouracil, capecitabine), topoisomerase I inhibitors (irinotecan, topotecan), poly (ADP-ribosidase) polymerase inhibitors (PARP) (e.g., olaparib), and the like. The pharmaceutical composition is selected according to the therapeutic requirements. These pharmaceutical compositions of the present invention can be administered in the form of tablets, capsules, oral preparations, powders, granules, tablets, liquid solutions for injection or infusion, suspensions, suppositories, inhalation preparations. See the following book for formulations: remington ("Remington: the science and Practice of medicine"), Lippincott Williams & Wilkins, 2000. One skilled in the art can select the form of administration, effective dosage, by screening for suitable diluents, adjuvants and/or excipients.
The term "pharmaceutical composition" refers to such forms of formulations: the active ingredient contained therein is biologically active and free of other ingredients having unacceptable toxicity to the individual to whom the formulation is to be administered. Another aspect of the invention is a method of producing the above antibody or a synthetic or recombinant fragment thereof, comprising the steps of: culturing the host cell, and purifying the antibody or synthetic or recombinant fragment thereof from the cell culture.
In the context of the present invention, the term "comprising" also includes the term "having predominantly" or "consisting essentially of … …".
In the present invention, "protein" as used herein also includes proteins, functional variants, functional derivatives, functional fragments or analogs thereof, isoforms, splice variants thereof encoded by the corresponding orthologous or homologous genes.
In the present invention, "functional" means, for example, "retaining its activity".
As used herein, a "fragment" refers to a polypeptide having a length of preferably at least 10 amino acids, more preferably at least 15, at least 17 or at least 20 amino acids, even more preferably at least 25 amino acids or at least 37 or 40 amino acids, and more preferably at least 50, or 100, or 150 or 200 or 250 or 300 or 350 or 400 or450 or 500 amino acids.
The invention is illustrated by reference to the non-limiting examples of the following figures.
FIG. 1: expression of IL-1R 8in human and mouse NK cells. a, b, expression of IL-1R8 protein in human primary NK cells and other leukocytes (a) and NK cell maturation stages (b). MFI, mean fluorescence intensity. c, d, IL-1r8mRNA expression in mouse primary NK cells and other leukocytes (c) and sorted spleen NK cell subsets (d). P <0.05, P <0.01, P <0.001, analysis of one-way variance (ANOVA). Mean ± s.e.m.
FIG. 2: NK cell differentiation and function in IL-1R8 deficient mice. a, b, Il1r8+/+And Il1r8-/-Frequency and absolute number of NK cells in leukocytes in mice. c, d, NK cell subgroup (c) and KLRG1+NK cells (d). e-g, IFN γ (e), telomerase B (f) and FasL (g) expression in stimulated NK cells. h, spleen CD27 after IL-18 depletion in vivoIs low inNK cell frequency. i, Il1r8+/+And Il1r8-/-NK cells in CpG-primed Il1r8+/+Dendritic cell co-culture and IFN γ production following IL-18 blockade. j, NK fineIRAK4, S6 and JNK phosphorylation following intracellular IL-18 stimulation. RNA-sequencing analysis in k, resting and IL-18 activated NK cells. Shows differentially expressed (P)<0.05) gene. l, correlation between IL-1R8 expression and IFN γ production in human peripheral blood NK cells. m, IL-1R8 expression and IFN γ production in duplicate 7 days after transfection of human NK cells with control siRNA or IL-1R8 specific siRNA. a-l, P between selected relevant comparisons<0.05,**P<0.01,***P<0.001, two-tailed unpaired student's t-test or mann-whitney U-test; k, r are Pearson's correlation coefficients. Mean ± s.e.m.
FIG. 3 NK cell mediated protection against liver carcinogenesis and metastasis in IL-1R8 deficient mice. a, Male Il1r8 after 6,8,10 and 12 months of DiethylNitro amine (DEN) injection+/+And Il1r8-/-Macroscopic scoring of liver lesions in mice the P-value, b, NK cell exudate frequency and representative tissue quantification (original magnification 20 ×; scale bar, 100 μm) c in the liver of tumor-bearing mice, IFN γ in the liver of tumor-bearing mice, is provided at the top of the graph+NK cell frequency. d, macroscopic scoring of liver lesions in male mice after NK cell depletion. e, number of spontaneous lung metastases. f, NK cell frequency in the lungs of MN/MCA1 tumor-bearing mice. g, number of lung metastases in MN/MCA1 tumor-bearing mice after NK cell depletion. h, number of liver metastases in MC38 colon tumor-bearing mice. i, j, Il1r8+/+Adoptive transfer of mice with Il1r8+/+And Il1r8-/-Number of lung (i) and liver (j) metastases after NK cells. a, d, representative images of female livers are shown. a-j, giving the exact P values between selected relevant comparisons, two-tailed unpaired student's t-test. Mean ± s.e.m.
FIG. 4 NK cell mediated antiviral resistance in IL-1R8 deficient mice. a, Il1r8+/+And Il1r8-/-Viral titers in the liver of infected mice. DL, detection limit. Day p.i., days post infection. b, IFN γ in infected mice+And CD107a+Frequency of NK cells. c, adoptive transfer of Il1r8+/+And Il1r8-/-Viral titers in neonatal wild-type mice after NK cells (7 days post infection). IFN γ in the liver of d, MCMV-infected mice+The frequency of the cells. a-d, giving exact P values, two-tailed Mann-Whitney U test (a, c) or unpaired student's t test(b, d). Median value (a, c); mean ± s.e.m. (b, d).
FIG. 5: expression of IL-1R 8in human and mouse NK cells. a, B, Il-1r8mRNA (a) expression in human primary NK cells, comparison with T and B cells, neutrophils, monocytes and in vitro derived macrophages (a), and in the maturation stage of human primary NK cells (CD 56)brCD16-,CD56brCD16+,CD56dimCD16+) And CD56dimCD16-Expression of subgroups (b). c, representative plots of fluorescence activated cell sorting for human NK cell subsets, and histograms of IL-1R8 expression in NK cell subsets. d, IL-1R8 protein expression in human bone marrow precursors and mature cells. e, ILR family member (Il1r1, Il1r2, Il1r3, Il1r4, Il1r5, Il1r6, Il1r8) mRNA expression in mouse primary NK cells isolated from spleen. f, expression of IL-1R8 protein in mouse NK cells as shown by confocal microscopy. Scale bar is enlarged, 10 μm. g, representative plot of fluorescence activated cell sorting of mouse NK cell subsets. a, b, d, P<0.05,**P<0.01,***P<0.001. One-way ANOVA. Mean ± s.e.m. a, n-6 (NK and B cells) or n-4 donors; b, n-5 donors; d, n-4 donors; e, n ═ 2 mice; f, representative images of 4 were collected per group. a, b, d-f, one experiment was performed.
FIG. 6: il1r8-/-Phenotypic analysis of NK cells. a, b, Il1r8+/+And Il1r8-/-Representative plot of fluorescence activated cell sorting of mouse NK cell subsets in mice (a) and histogram of KLRG1 expression in NK cells (b). c, d, Il1r8+/+And Il1r8-/-Absolute number of NK and NK cell subsets (DN, CD11 b) in bone marrow, spleen and blood of newborn mice at 2(c) and 3(d) weeks of ageIs low inDP and CD27Is low in)。e,Il1r8+/+And Il1r8-/-Frequency of bone marrow precursors in mice. f, Il1r8+/+And Il1r8-/-NKG2D, DNAM-1 and LY49H expression of peripheral NK cells and NK cell subsets in mice. g, Il1r8+/+And Il1r8-/-Splenic perforin post-stimulation in mice+NK cell subgroup frequency. h, i, absolute number of peripheral NK cells (h) and CD27 in bone marrow chimeric mice after reconstitution (9 weeks)Is low inNK cell frequency (i). j, k, transplanted with 50% Il1r8+/+CD45.1 cells and 50% Il1r8-/-Competent chimeric mice of CD45.2 cells had peripheral NK cell (j) and NK cell subset (k) frequencies after reconstitution (9 weeks). Following reconstitution, Il1r8 was observed under competent conditions for defective implantation (12% rather than 50% implantation)-/-A stem cell. l, Il1r8+/+And Il1r8-/-NK cells in LPS-or CpG-primed Il1r8+/+And Il1r8-/-IFN γ production following dendritic cell co-culture. c-l, P between selected relevant comparisons<0.05,**P<0.01,***P<0.001, two-tailed unpaired student's t-test. Median and error bars, mean ± s.e.m. At least five animals per group were used. c, d, three merging experiments; e-l one experiment was performed.
FIG. 7: NK cells IL-1R8 dependent regulatory mechanism. a, in wild type, Il1r8-/-,Il18-/-And Il18-/-/Il1r8-/-Spleen CD27 in miceIs low inNK cell frequency. b in wild type, Il1r8-/-,Il1r1-/-And Il1r8-/-Il1r1-/-Peripheral CD27 in mouse (left side)Is low inNK cell frequency and IFN γ production by splenic NK cells following IL-12 and IL-1 β or IL-1 β stimulation (right)+/+And Il1r8-/-Mice were depleted of commensal flora (c) and co-fed conditions (d) spleen CD27Is low inNK cell frequency. e, STED microscopy of IL-18 stimulated human NK cells. Scale bar, 2 μm. a-d, P between selected correlation comparisons<0.05,**P<0.01,***P<0.001, two-tailed unpaired student's t test; median and error bars, mean ± s.e.m. a, n-3, 5, or6 mice; at least 5 animals per group (b-d) were used. a-d, one experiment was performed. e, three representative images collected from two donors.
FIG. 8: il1r8+/+And Il1r8-/-RNA sequencing analysis of NK cells. Resting and IL-18 activated Il1r8+/+And Il1r8-/-Metascape analysis of NK cell enrichment Gene pathway. See also the data stored in NCBI gene expression synthesis database accession number GSE 105043.
FIG. 9: NK cell mediated resistance to hepatocellular carcinoma and metastasis in IL-1R8 deficient mice. a, female Il1r8+/+And Il1r8-/-Mice were macroscopically scored for liver lesions at 6, 10 and 12 months after Diethylnitramine (DEN) injection. b, Il1r8+/+And Il1r8-/-Incidence of hepatocellular carcinoma in female and male mice. c, Il1r8+/+And Il1r8-/-IFN gamma in spleen of tumor-bearing mice+NK cell frequency. d, female Il1r8+/+And Il1r8-/-Macroscopic scoring of liver foci in mice following NK cell depletion. e, Il1r8 after NK cell depletion+/+And Il1r8-/-Quantification of 2-deoxyglucosone (2-DG) in the lung of tumor-bearing mice. f, Il1r8+/+And Il1r8-/-Primary tumors in mice (25 days after MN/MCA1 cell line injection) grew. g, Il1r8+/+And Il1r8-/-MN/MCA1 Holland sarcoma mice in IFN gamma or IL-18 after neutralization of lung metastasis number. h, Il1r8+/+And Il1r8-/-MN/MCA 1-tumor bearing mice in IL-17A or CD4+/CD8+Lung volume was transferred after cell depletion. i, Il1r8+/+And Il1r8-/-,Il1r1-/-,Il1r1-/-/Il1r8-/-MN/MCA 1-number of lung metastases in mice carrying them. j, Il1r8+/+,Il1r8-/-,Il18-/-,Il18-/-Il1r8-/-Number of liver metastases in MC38 colon cancer-bearing mice. k, Il1r 83 or7 days after adoptive transfer+/+And Il1r8-/-Absolute number of NK cells. l, in vivo Il1r 83 days after adoptive transfer+/+And Il1r8-/-NK cells are expanded. m, Il1r 8in adoptive transfer+/+And Il1r8-/-NK cells with PMA-enomycin, IL-12 and IL-18 stimulation after 4 hours of isolated IFN gamma production and degranulation. n, Il1r8+/+Adoptive transfer of IL1r 8in MN/MCA1 tumor-bearing mice+/+And Il1r8-/-Lung metastasis volume after NK cells. a, c-e, g-j, m-n, P between selected relevant comparisons<0.05,**P<0.01,***P<0.001, two-tailed unpaired student's t-test or mann-whitney U-test. # P<0.05,##P<0.01, Kruskal-Wallis and Dunn's multiple comparison test. Median and error bars, mean ± s.e.m.a, n ═ 9,10,11,18,21 mice; b, n-8-21 mice; c, n ═ 6 mice; d, n ═ 10,12,13 mice; e, n ═ 4(Il1r 8)-/-Isoform) or n ═ 5; f, n is 10; g, n ═ 6,7,9,10 mice; h, n-5, 6,12 mice(ii) a i, n ═ 6,8,10 mice; j, n ═ 4,5,7 mice; k, l, m, n ═ 3 mice; n, n ═ 9,10,12 mice. Representative experiments are those from three (a, b),2(d),6(f), or one (c, e, g-n) run.
FIG. 10: NK cell mediated antiviral resistance in IL-1R8 deficient mice. Il1r8+/+And Il1r8-/-Cytokine serum levels in infected mice (1.5 and 4.5 days (p.i.) post infection). P<0.05,**P<0.01,***P<0.001, unpaired student's t-test. Median and error bars, mean ± s.e.m.; n-5 mice. An experiment was performed.
FIG. 11: mouse spleen NK cell gating strategy for FACS analysis and NK cell sorting.
FIG. 12: NK cells are functionally activated by anti-PD-1. NK cells were activated under baseline conditions (incubated in the presence of control antibody (CTRL) alone) or in culture with target (stimulated MC38 colorectal cancer cells) and anti-PD-1 antibody (aPD-1), staining intracellularly for IFN γ (upper panel) and telomerase B (lower panel). NK cells were purified and processed as described in the methods section and analyzed by flow cytometry. MFI-mean fluorescence intensity. Student's t test. N-2 mice.
FIG. 13: IL-1R8 expression in human lymphocytes. IL-1R8 expression was analyzed by flow cytometry. CD8+ T cell subsets were determined based on the following gating strategy: a) naive T cell subsets: CD3+, CD8+, CCR7+, CD45RO-, b) Stem Cell Memory (SCM) T cell subsets: CD3+, CD8+, CCR7+, CD45RO-, CD95 +; c) effector T cell subsets: CD3+, CD8+, CCR7-, CD45RO +; d) terminal effector T cell subset: CCR7-, CD45 RO-; central memory (Mem): CD3+, CD8+, CCR7+, CD45RO +. MFI-mean fluorescence intensity.
FIG. 14: mouse CD8+ T cells proliferate and mature. A) CD8+ T cell proliferation was assessed as described above in the methods and the percentage of dividing cells was reported. B) Expression of the mature marker CD44 upon activation. Student's t test. N ═ 6 mice.
FIG. 15: CD8+ T cell activation. Expression of IFN γ (A, B) and telomerase B (C, D) following stimulation with anti-CD 3/CD28 and cytokines (IL-2, IL-12, IL-18). Results report the percentage of positive cells or Mean Fluorescence Intensity (MFI). Student's t test. N-4 mice.
TABLE 1 serum cytokine and liver enzyme levels in mice bearing hepatocellular carcinoma
Figure BDA0002550531970000201
Figure BDA0002550531970000211
Samples without detectable levels were not included in the assay.
Levels are U/L.
n- DEN 5 or 8 months after
Example 1
Materials and methods
Animal(s) production
The genetic background for all female and male mice used was C57BL/6J, 8-12 weeks old, unless otherwise indicated. Wild type mice were obtained from Charles River Laboratories, Ill 1r8/Litter size in mice. IL-1R 8-deficient mice produced as described31。Il1r1/Mice were purchased from Jackson Laboratory, Berkson Laboratory, Burdock, Myelina, USA (The Jackson Laboratory). All colonies were housed in individual ventilated cages at the SPF animal facility at the Humanitas clinical research center. By hybridization of Il1r1/And Il1r8/Mouse production of Il1r1//Il1r8/A mouse. By hybridization Il18/And Il1r8/Mouse production of Il18//Il1r8/A mouse. Mice were randomly grouped based on gender, age, and weight. Procedures involving animal handling and care were in accordance with protocols approved by the Humanitas clinical research center (roxzano, Milan, Italy), consistent with national (d.l.n.116, g.u., suppl.40,18-2-1992 and n.26, g.u.2014, 3 months and 4 days) and international laws and guidelines (EEC committee directive 2010/63/EU, OJ L276/33, 22-09-2010; national health council for health guidelines for experimental animal care and use, american national research council, 2011). The study was approved by the Italian department of health (Standard letters 43/2012-B, 2.2012, 8.d.)Hair, and 828/2015-PR number, issued 8/7/2015). The best efforts are made to reduce the number of animals used and their suffering. In most in vivo experiments, the investigators do not know the genotype of the experimental group.
Human primary cells
Human peripheral blood mononuclear cells were isolated from peripheral blood of healthy donors according to approval by the ethical committee of the human manitas research hospital. Peripheral blood mononuclear cells were obtained by Ficoll density gradient centrifugation (GE healthcare biosciences). NK cells were then purified by negative selection using magnetic cell sorting technology (EasySep human NK cell enrichment kit, stem cell technology) according to the protocol provided by the manufacturer. Human monocytes were obtained from peripheral blood of healthy donors by two-step gradient centrifugation, first Ficoll and then Percoll (65% isotonic; Pharmacia, Uppsala, Sweden). Residual T and B cells were removed from the monocyte fraction by plastic adhesion. In a medium supplemented with 10% Fetal Bovine Serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin and 100ng ml-1Monocytes were cultured for 7 days in RPMI-1640 medium of M-CSF (Peprotech) to produce resting macrophages. T and B cells were obtained from peripheral blood of healthy donors using RosetteSep human T cell enrichment mixtures and RosetteSep human B cell enrichment mixtures (stem cell technology) according to the manufacturer's instructions. Neutrophils were enriched from Ficoll-isolated granulocytes using the EasySep human neutrophil enrichment kit (stem cell technologies) according to the manufacturer's instructions. To analyze pluripotent hematopoietic stem cells and NK cell precursors, human bone marrow mononuclear cells were collected from Humanitas Biobank approved by the Humanitas research Hospital ethical Committee (grant No. 1516,2016, 2.26). Frozen samples were thawed and activity assessed using trypan blue and Aqua LIVE/Dead-405nm staining (Invitrogen) and used for flow cytometry analysis. Informed consent was obtained from all participants.
Fluorescence activated cell sorting analysis
Single cell suspensions of bone marrow, blood, spleen, lung and liver were obtained and stained. Figure 11A shows a representative NK cell gating strategy. Buffer set stained with Foxp 3/transcription factor (eBio)science) intracellular staining for telomerase B and perforin. Intracellular staining of IFN with Cytofix/Cytoperm (BD biosciences). Liver ILC1 was identified as NK1.1+CD3-CD49a+CD49b-Cells with intracellular staining IRAK, pIRAK, pS and JNK with formaldehyde 4% and methanol 100%, using mouse antibodies CD-BV 605, -BV650 or-PerCp-Cy5.5 (clone 30-F), CD45.1-BV650 (clone A), CD45.2-APC, -BV421 (clone 104), CD 3-PerCP-Cy5.5 or-APC (clone 145-2C), CD-PerCP-Cy5.5, -eFluor450 (clone 1D), NK1.1-PE, -APC, -eFluor450 or-biotin (clone PK136), CD 11-BV 421, -BV450, -BV785 (clone M/70), CD-APC-Fluor 780 (clone 7F9), CD-FITC (clone RM-5), CD-PE (clone 53-6.7), KL-1-BV (clone 2F), CD-2-AFE 780 (clone 201-APC-7), CD 111-APC-7-AFE (clone 201-7), CD-APC-AFE-11-7 (CD-7-APC-AFE), CD-11-EPO-11-7 (CD-EPO-7, CD-EPO-11-7, CD-EPO-11-7 (CD-EPO-14), CD-EPO-14-EPO-Bt-11, CD-11-Bg), CD-11-Bg-11-Bt-11-Bt, CD-11 (Bt, CD-11-Bt, CD-11-Bt, CD-11-Bt, CD-11, CD-11-Bt, CD-11, CD-7 (Bt, CD-11, CD-7, CD-Bt, CD-7, CD-11, CD-7, CD-11, CD-7, CD-&D Systems)) and streptavidin Alexa Fluor647 (Invitrogen) human NKT cells were detected with PE-CD1D tetramer (ProImmune, Oxford, England) loaded with α GalCer using the antibodies for detection of protein phosphorylation p-IRAK4 Thr345/Ser346 (clone D6D7), IRAK4, p-S6-Alexa647Ser235/236 (clone D57)2.2E); p-SAPK/JNK Thr183/Tyr185 (clone 81E11), from Cell signalling Technology. p-IRAK4, IRAK4 and p-SAPK/JNK were stained with goat anti-rabbit Alexa Fluor647 secondary antibody (Invitrogen). Results reported the mean fluorescence intensity normalized to isotype control or fluorescence subtraction (fluorogenic one). Cell viability was determined by Aqua LIVE/Dead-405nm staining (Invitrogen) or by the fixable reactive dye (FVD) eFluor780 (eBioscience); negative cells were considered viable. Cells were analyzed on LSRFortessa or FACSVerse (BD biosciences). Data were analyzed using FlowJo software (Treestar).
Quantitative PCR
Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's recommendations. The RNA was further purified using the miRNeasy RNA isolation kit (Qiagen) or Direct-zol RNA MiniPrep Plus (Zymo research). cDNA was synthesized by reverse transcription using a high-throughput cDNA Archive kit (Applied Biosystems), and quantitative real-time PCR was performed in a CFX96 Touch real-time PCR detection system (Bio-Rad) using a SybrGreen PCR master mix (Applied Biosystems). PCR reactions were performed with 10ng of DNA. By 2(-ΔCT)Data were analyzed by methods based on GAPDH, β -actin or 18S expression determined with the same sample, data were normalized as shown
Figure BDA0002550531970000241
Figure BDA0002550531970000251
Mouse leukocyte purification
By passing
Figure BDA0002550531970000252
Spleen NK cells and bone marrow neutrophils were enriched according to the manufacturer's instructions (american and whirlpool biotechnology). Such as fluorescence activated cell sortingThe purity of NK cells was determined to be about 90%. The purity of the neutrophil is more than or equal to 97.5 percent. Staining NK cells (CD45-BV650, NK1.1-PE, CD3e-APC, CD11b-BV421, CD27-FITC) and sorting on a FACSAria cell sorter (BD biosciences) to obtain high-purity NK cells and NK cell populations (CD11 b)Is low inCD27Is low in,CD11bIs low inCD27Height of,CD11bHeight ofCD27Height ofAnd CD11bHeight ofCD27Is low in). Spleen B and T lymphocytes were stained (CD45-PerCP, CD3e-APC, CD4-FITC, CD8-PE, CD19-eFluor450) and sorted. The purity of each population is more than or equal to 98 percent. The resulting cells are processed for mRNA extraction or for adoptive transfer, or for co-culture experiments. In vitro derived macrophages were obtained from bone marrow whole cells. In a medium supplemented with 10% Fetal Bovine Serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin and 100ngml-1Bone marrow cells were cultured in RPMI-1640 medium of M-CSF (Peprotech) for 7 days to produce resting macrophages. In a medium supplemented with 10% Fetal Bovine Serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin and 20ng ml-1Bone marrow cells were cultured in RPMI-1640 medium of M-CSF (Peprotech) for 7 days to produce dendritic cells.
Confocal microscopy analysis
Mouse splenic NK cells were enriched by magnetic cell sorting, allowed to adhere to poly-D-lysine (Sigma Aldrich) coated cover glass, fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and incubated with blocking buffer (5% normal donkey serum (Sigma Aldrich), 2% BSA, 0.05% Tween). The biotin-conjugated goat polyclonal anti-SIGIRR antibody or biotin-conjugated standard goat IgG was then used as a control (both from RD systems) (10. mu.g ml)-1) Staining was then performed with Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (Molecular Probes) and 4', 6-diamidino-2 phenylindole (DAPI) (Invitrogen). Anti-fade media fluoroPreserve reagent (EMD Millipore) was applied to coverslips and analyzed with an Olympus FluoviewFV1000 laser scanning confocal microscope (equipped with a 40-fold oil lens (numerical aperture 1.3)).
Stimulated depletion (STED) microscopy
Human cells were enriched and adhered by poly-D-lysine (Sigma Aldrich) coating on coverslips and IL-18(50ng ml)-11 min, 5 min, 10 min) stimulation, fixation with 4% PFA, incubation with 5% normal donkey serum (Sigma Aldrich), 2% BSA, 0.05% Tween in PBS2+ (pH 7.4) (blocking buffer), followed by incubation with biotin-conjugated goat polyclonal anti-human IL-1R8 antibody or biotin-conjugated normal goat IgG (all from RD systems) and mouse monoclonal anti-IL-18R α (clone 70625; RD systems) or mouse IgG1 (Invitrogen), all at 5 μ g ml-1Diluted in blocking buffer and then incubated with Alexa Fluor 488-conjugated donkey anti-goat IgG antibody and Alexa Fluor555 donkey anti-mouse IgG antibody (both from molecular probes company.) steamxz images were obtained in a unidirectional mode with leka SP8 STED3X confocal microscope system.alexa Fluor 488 was excited with 488nm argon laser, rays were collected at 505 and 550nm, gating between 0.4 and 7ns was used to avoid reflections and autofluorescence.alexa Fluor555 was excited with white laser adjusted to 555/547nm, emission at 580 and 620nm was collected.linear order acquisition was used to avoid fluorescence overlap.for two excitations with 660nm CW-attenuated laser (80% power.) Alexa PLAPO 100 ×/1.40 numerical STED white oil was collected at 572.3 milli absorption units (mAU) using professional steaxa software for Alexa Fluor555 and Alexa Fluor555 acquisition.
3' -mRNA sequencing and analysis
Splenic NK cells (six mice from each genotype, pooled in pairs) were purified as described above and stimulated with IL-18(MBL) (20ng ml)-14 hours). RNA was prepared as described above. The QuantSeq 3' mRNA-sequencing library preparation kit Illumina (Lexogen) was used to generate libraries for sequencing on NextSeq (Illumina; 75bp PE). The fastq sequence file was evaluated using the fastqc program. Firstly, the bbduk is used for sleeving bbmap software32Reads were trimmed, the first 12 bases removed, and contaminants were removed as found by contaminant kmer (length 13). An area having a length of 20 or more and an average mass of less than 10 was cut out from the end of the reading. However, the device is not suitable for use in a kitchenPost-crop reading, using cutadapt33The tailed poly G and poly a readings were removed and the quality of the remaining readings was re-evaluated with fastqc. The cut reads were aligned to the mm10 genome reference using the STAR program34The readings are assigned to features in the mm10 annotation. Differential expression analysis R/bioconductor calibration with TMM35edgeR software package36The normalized linear model function of (1). Genome analysis Using the R/bioconductor software package limma38Middle romer37And (4) performing functions. The genes were enriched with Metascape (http:// Metascape. org) for genetic cancer biology procedures, KEGG Pathway and Pathway and Reactome gene sets.
Cytokine assay
Cytokines were measured using a BD flow bead array (CBA) mouse inflammation kit (BD) or a Duoset ELISA kit (RD systems Co.).
In vitro functional assay
Whole mouse spleen cells or enriched mouse or human NK cells were cultured in RPMI-1640 medium supplemented with 10% FBS 1% L-glutamine, 1% penicillin/streptavidin, and specifically treated with IL-2, IL-12, IL-15(Peprotech), IL-18(MBL), IL-1(Peprotech) and PMA-enomycin (Sigma Aldrich). With IL-18(50ng ml)-1),IL-15(50ng ml-1),IL-2(20ng ml-1) And IL-12(10ng ml)-1) FasL expression was assessed 45 minutes after treatment. With IL-12(20ng ml)-1) And IL-18(20ng ml)-1) Or IL-1(20ng ml)-1) After 16 hours of treatment, IFN γ production was analyzed by using BD Cytofix/Cytoperm fixation/permeabilization kit according to the manufacturer's instructions or by ELISA intracellular staining. Intracellular staining with telomerase B and perforin was performed by using IL-12(10ng ml)-1),IL-15(10ng ml-1) And IL-18(50ng ml)-1-1) After 18 hours of stimulation, the cells were incubated with Foxp 3/transcription factor staining buffer set (eBioscience). The CD107a-Alexa Fluor647 antibody was added at 4 hours of culture and analyzed by flow cytometry. BD GolgiPlug (containing brefeldin) and BD GolgiStop (containing monensin) were added 4 hours prior to intracellular staining. When indicated, PMA (50ng ml) was added 4 hours before intracellular staining-1) And enomycin (1. mu.g ml)-1)。
NK-dendritic cell coculture experiments were performed as described above39. Using LPS (Sigma Aldrich; 1. mu.g ml) from Escherichia coli (Escherichia coli) O55: B5-1) Or CpG ODN 1826 (invitrogen; 3 μ g ml-1) anti-mIL-18 neutralizing antibodies (BioXCell, clone YIGIF74-1G 7; 5 μ g ml-1) Or rat isotype control (BioXCell, clone 2a 3).
After viral infection, flow cytometry was performed on BD GolgiPlug, BD GolgiStop and IL-2(500U ml)-1) IFN and CD107a expression were analyzed 4 hours after treatment.
With IL-18(10ng ml)-1) Phosphorylation of IRAK4, S6 and JNK was analyzed 15-30 min after stimulation.
Transfection of human primary NK cells
Human NK cells were enriched from peripheral blood of healthy donors and transfected with Dharmacon Acell siRNA (GE healthcare) using Accell transfer medium (GE healthcare) according to the manufacturer's instructions. SIGIRR-specific siRNA (1. mu.M) (On-Target Plus; Dharmacon, GE healthcare) included 250nM of the following four antisense sequences: i, AGU UUC GCGAGC CGA GAU CUU (SEQ ID NO: 1); II, UAC CAG AGC ACG UUG AUU (SEQ ID NO: 2); III UGA CCC AGG AGU ACU CGU GUU (SEQ ID NO: 3); IV, CUU CCC GUC GUU UAU CUC CUU (SEQ ID NO:4) (all 5 to).
Production of bone marrow chimeras
Lethal irradiation of Il1r8 with a total radiation dose of 900cGy/And Il1r8+/+Two hours later, the mice were injected with 4 × 10 at the posterior orbital plexus6Nucleated bone marrow cells obtained by washing wild type or Il1r8-/-The donor was obtained from freshly resected leg bone cavity. By using 50% CD45.1 Il1r8+/+And 50% CD45.2 Il1r8/Bone marrow cell reconstitution recipient mice generated competent bone marrow chimeric mice. From 10 days before irradiation to two weeks after irradiation, recipient mice received gentamicin (0.8mg ml)-1Dissolved in drinking water). NK cells of the chimeric mice were analyzed 8 weeks after bone marrow transplantation.
Exhaustion and occlusion experiments
Mice were treated intraperitoneally with 200 μ G of specific mAb (mouse anti-NK 1.1, clone PK 136; mouse isotype control, clone C1.18.4; rat anti-mIL-18, clone YIGIF74-1G 7; rat isotype control, clone 2A 3; rat anti-IFN, clone XMG 1.2; rat IgG1 HRPN; mouse anti-IL-17A, clone 17F 3; mouse isotype control, clone MOPC-21; rat anti-CD 4/CD8, clone GK 1.5/YTS; rat isotype control, clone LTF-2 (all from BioXCell)) and then with 100 μ G (anti-NK 1.1) or three times (anti-IL-18, anti-IFN, anti-IL-17A, anti-CD 4/CD8) once a week for the duration of the experiment.
Exhaustion of microbial groups
10mg ml of an antibiotic cocktail (ampicillin (Pfizer)) were orally administered by tube daily-1Vancomycin (PharmaTech Italiaa, Italian technology) 10mg ml-1Metronidazole (Societ-Prodotti antibiotics Co.) 5mg ml-1And neomycin (Sigma Aldrich) 10mg ml-1) Six week old mice were treated for 5 weeks. Control mice were treated with drinking water. The feeding volume of 10ml/kg (body weight) was transferred by stainless steel tube without previously calming the mice. DNA was isolated from bacterial fecal pellets using a PowerSoilDNA isolation kit (MO BIO laboratories Inc.) and quantified spectrophotometrically at 260 nm. PCR was performed with 10ng of DNA using a SybrGreen PCR master mix (applied biosystems) in a CFX96 Touch real-time PCR detection System (Burley). By 2(-ΔCT)Methods (applied biosystems, guide to real-time PCR applications) the data were analyzed.
Cancer model
At 15 days of age, mice were injected intraperitoneally with 25mg/kg (body weight) of diethylnitramine (sigma). 6. After 8,10 or 12 months, the liver cancer was analyzed by euthanasia. Liver cancer scoring was based on the number and volume of lesions (0: no lesions; 1: number of lesions)<3, or lesion diameter<3 mm; 2 number of lesions<5, or lesion diameter<5 mm; number of lesions<10, or lesion diameter<10 mm; 4 number of lesions<15, or lesion diameter<10 mm; 5 number of lesions>15, or lesion diameter>10 mm). Intramuscular injection of the 3-MCA-derived Mycoplasma-free sarcoma cell line MN/MCA1 (10)5Individual cells/mouse in 100 μ l PBS) for lung metastasis experiments40. Primary tumor growth was monitored twice a week by in vivoLung metastasis was assessed by imaging and macroscopic enumeration at 25 days euthanasia post-injection.intrasplenic injection 1.5 × 105A Mycoplasma free colon cancer cell (MC38)21Producing liver metastases. Mice were euthanized 12 days after injection and liver metastases were counted macroscopically. MC38 cells were obtained from ATCC just prior to use. MN/MCA1 cells were morphologically verified in vitro by microscopy and ex vivo by histology. The tumor size limit at euthanasia of mice is based on the major diameter (not more than 2 cm).
Viral infection
Mice were injected intravenously with 5 × 10 in PBS5Plaque forming units of tissue-cultured virus. Bacterial artificial chromosome-derived MCMV strain MW97.01, previously shown to be biologically equivalent to MCMV strain Smith (VR-1399), and is referred to herein as wild-type MCMV41. 1.5 and 4.5 days after infection, mice were euthanized and plaque assays were performed as described above42,43Viral titers were evaluated. Neonatal mice were intraperitoneally infected with 2,000 plaque forming units of MCMV strain MW97.01 and euthanized 7 days post infection. As described above42,43Viral titers were assessed using plaque assays.
Adoptive transfer
Intravenous injection 10 in wild type adult mice6II 1r8+/+Or Il1r8/Sorted NK cells were injected 5 hours later with either MN/MCA or MC38, or intravenously into neonatal mice 48 hours after MCMV injection. Adoptively transferred NK cell implants, proliferation capacity and functionality (degranulation and IFN production after ex vivo stimulation) were evaluated 3 and 7 days after injection.
Proliferation in vivo
In vivo proliferation was measured using the Click-iT Edu flow cytometry assay kit (Invitrogen). Edu (0.5 mg/mouse) was injected intraperitoneally, and the mice were euthanized 24 hours later, stained for cells according to the manufacturer's instructions, and analyzed by flow cytometry.
Immunohistochemistry
Frozen liver tissue was cut into 8mm size and fixed with 4% PFA. With 0.03% H2O2Endogenous peroxidase was blocked for 5 min and non-specific binding sites were blocked with PBS + 1% FBS for 1 h. Polyclonal goat anti-mouse NKp46/NCR1(RD line) for tissueSystemcomp) staining, goat-versus rodent HRP polymer kit (GHP516, Biocare Medical) was used as secondary antibody. The reaction was developed with 3, 3' -diaminobenzidine (Biocare Medical) and the slides were counterstained with hematoxylin. Slides were mounted with eukitt (sigma aldrich). Images were analyzed at 20-fold magnification using cell ^ F software (Olympus).
In vivo imaging
After raising with AIN-76A alfalfa free diet (Mucedola, italy) for two weeks to reduce the fluorescence background, mice were injected intravenously with XenoLight red 2-deoxyglucoside (PerkinElmer), and 24 hours later the 2-deoxyglucoside fluorescence was measured with a fluorescence molecular tomography system (FMT2000, PerkinElmer). The images obtained were then analyzed with TrueQuant 3.1 analysis software (perkin elmer).
Statistical analysis
For animal studies, sample sizes were determined based on past experience with cancer and infection models to detect differences between groups of 20% or more (10% significant levels and 80% efficacy). Values are expressed as the mean ± s.e.m, or median, of the biological replicates, as specified. Multiple groups were compared using a one-way ANOVA or Kruskal-Wallis test. The unmatched groups were compared to the gaussian distribution using a two-sided unpaired student's t-test, using Welch's correction for examples with significantly different variances. For the case of no gaussian distribution, the mann-whitney U test is used. Outliers were excluded by ROUT test. P.ltoreq.0.05 was considered significant. Statistics were calculated using GraphPad Prism version 6, GraphPad software.
Statistics and replication
Figure 1a, n-4 (B-cells), n-5 (NKT-cells), n-9 (T-cells), n-10 (NK-cells) donors; figure 1b, n-5 donors; figure 1c, n-8 (NK cells) or n-4 (T cells) or n-3 (other leukocytes) mice; fig. 1d, n-5 mice. Figure 1b, representative of six experiments performed. Fig. 1a, c, d, an experiment was performed.
Fig. 2a, b, n-8 or n-7 (spleen, Il1r8)+/+Liver) or n ═ 6(Il1r 8)/Liver) mice; fig. 2c, n ═ 6 mice; fig. 2d, n ═ 9(Il1r 8)+/+) Or n is 6(Il1r8/) Only mice; figure 2e, n-5 mice; fig. 2f, n ═ 6 mice; figure 2g, n-4 mice; fig. 2h, n ═ 5 mice; fig. 2i, n ═ 10 pores; fig. 2j, n-4 (IRAK4), n-6 or n-5 (S6Il1r 8)/) Or n-7 (JNK Il1r8)/) Only mice; fig. 2k, n ═ 3 mice; figure 2l, n ═ 9 healthy donors; figure 2m, n-4 healthy donors. Representative of three (fig. 2a, b), five (fig. 2c), two (fig. 2d, j), four (fig. 2e) experiments performed. FIGS. 2f-m, one experiment was performed.
Fig. 3a, n-8, 10,11,13,14 mice; fig. 3b, c, n ═ 6 mice; fig. 3d, n-10, 12,13 mice; fig. 3e, n-10, 11 mice; fig. 3f, n-5, 6,7 mice; fig. 3g, n-9, 10 mice; fig. 3h, n-5, 6 mice; figure 3i, n-9, 10 or 12 mice; fig. 3j, n ═ 6 mice. Representative experiments in runs 6 (FIG. 3e),3 (FIG. 3a),2 (FIG. 3d, f, g, h, i). Fig. 3b, c, j, an experiment was performed.
Fig. 4a, b, n-5 mice; fig. 4c, n-6, n-9 mice; fig. 4d, n ═ 4 mice. Fig. 4a, two experiments were performed. Fig. 4b-d, one experiment was performed.
Results
IL-1R8 is widely expressed10. However, the inventors found that human NK cells had much higher levels of IL-1R8mRNA and protein in comparison to other circulating leukocytes and monocyte-derived macrophages (fig. 1a and 5 a). IL1R8mRNA levels are elevated during NK cell maturation11(FIG. 5b), surface protein expression reflects the level of transcription (FIGS. 1b and 5 c). Low levels of IL-1R8 expression were detected in bone marrow pluripotent hematopoietic stem cells and NK cell precursors, and were selectively upregulated in mature NK cells, but not in CD3+ lymphocytes (fig. 5 d).
Mouse NK cells expressed significantly higher levels of Il1r8mRNA compared to other leukocytes (fig. 1c) and other ILRs (fig. 5e, f). Consistent with the results obtained with human NK cells, Il1r8mRNA levels were at levels from CD11bIs low inCD27Is low inTo CD11bHeight ofCD27Is low in(literature)12) Increased four-stage developmental transition (fig. 1d and fig. 5 g).
To assess the role of IL-1R 8in NK cells, the inventors utilized IL-1R8 deficiencyA type mouse. In CD45+In the cells, the frequency and the absolute number of NK cells are Il1r8/The peripheral blood of the mouse is obviously higher than Il1r8+/+Mice, and slightly increased in the liver and pancreas. (FIGS. 2a, b). In addition, CD11bHeight ofCD27Is low inAnd the KLRG1+ mature subgroup at Il1r8/The bone marrow, spleen and blood of the mouse are obviously higher than Il1r8+/+Mice, demonstrating a more mature NK cell phenotype13(FIGS. 2c, d and FIGS. 6a, b).
Il1r8/Enhanced NK cell maturation has occurred in mice at 2 and 3 weeks of age, while the frequency of NK precursors was in Il1r8/And Il1r8+/+Similar in bone marrow, indicating that IL-1R8 regulates early events in NK cell differentiation without affecting NK cell precursor development12(FIGS. 6 c-e).
The inventors next investigated whether IL-1R8 affects NK cell function. Expression of activating receptors NKG2D, DNAM-1 and Ly49H in peripheral blood Il1r8/Significant up-regulation in NK cells (fig. 6 f). IFN-. gamma.interferon (IFN and granzyme B production and FasL expression are more persistent after ex vivo stimulation in IL-1R8 deficient NK cells in the presence of IL-18 (FIGS. 2 e-g and 6g)+NK cells in Il1r8/The frequency was higher in total NK cells and all NK cell subsets. Thus, the enhancement of IFN production is independent of the state of cell maturation. Analysis of competent bone marrow chimeras revealed that IL-1R8 regulated NK cell differentiation in a cell-autonomous manner (FIG. 6 h-k). Similarly, NK cell coculture experiments with Lipopolysaccharide (LPS) or CpG-primed dendritic cells showed Il1r8/NK cells were not restricted by dendritic cell genotype, producing higher IFN levels (fig. 6 l).
IL-18 is an IL-1family member that plays an important role in NK cell differentiation and function1,14. Due to IL-18 blockade or gene defect, Il1r8/Enhanced NK cell maturation and effector function in mice was abolished but not affected by IL-1R1 deficiency (fig. 2h, i and fig. 7a, b). Co-feeding and antibiotic treatment had no effect, thus excluding Il1r8/Small and medium-sized biota of mice15Effect on phenotype (fig. 7c, d).
The results reported above suggest that,IL-1R8 regulates the IL-18 signaling pathway in NK cells; indeed, IL-18 is at Il1r8 compared to wild type NK cells/The induced increase in phospho-IRAK 4/IRAK4 ratio in NK cells indicates early signaling release downstream of MyD88 and formation of Mydd bodies (Myddosomes) (FIG. 2j), consistent with the putative molecular model of IL-1R8 action (documents 1,9, 16). Indeed, the inventors observed that IL-1R8 and IL-18R clustered by stimulated depletion (STED) microscopy (FIG. 7e), consistent with previous studies9. IL-1R 8-deficiency also resulted in increased IL-18 dependent phosphorylation of S6 and JNK in NK cells, suggesting that IL-1R8 inhibits IL-18 dependent activation of mTOR and JNK pathways (FIG. 2j), which controls NK cell mechanisms, differentiation and activation17,18。
To gain a deeper understanding of the effects of IL-18 dependence on NK cell function and on IL-18 response, RNA sequencing (RNA-seq) analysis was performed. IL-1R8 deficiency has a significant effect on the resting transcriptional profile of NK cells, particularly on the responsiveness to IL-18 (FIG. 2k, FIG. 8a, data saved at NCBI Gene expression integration database under accession number GSE 105043). The profile of IL-1R 8-deficient cells includes increased production of activation pathways (e.g., MAPK), associated adhesion molecules in cell-cell interactions and cytotoxicity (ICAM-1), and selected chemokines (CCL 4). The end of these data represents NK cell-based leukocyte recruitment, including the amplification loop of the NK cells themselves.
To investigate the role of IL-1R 8in human NK cells (FIGS. 1a, b), the inventors first analyzed in reverse the expression associated with the response of IL-18 and IL-12 combinations in their normal donors. The inventors observed that IL-1R8 levels and IFN production are inversely correlated in peripheral blood cells (R)20.7969, P0.0012) (fig. 2 l). Furthermore, partial silencing of IL-1R8 by small interfering rna (sirna) in peripheral blood NK cells was associated with a significant increase in IFN production (fig. 2m) and upregulation of CD69 expression (data not shown). These results indicate that in human NK cells, as in the mouse counterpart, IL-1R8 acts as a negative regulator of activation, whose inactivation releases effector functions of human NK cells.
To assess the actual relevance of IL-1R 8-mediated NK cell regulation, anti-cancer and antiviral resistance were examined. High in liverFrequency of NK cells19. Therefore, the inventors focused on liver cancer development. IL-1R 8-deficient male and female mice in a diethylnitramine-induced hepatocellular carcinoma model20Protection from foci was determined based on macroscopic number, size (fig. 3a and fig. 9a, b) and histology (data not shown). Percentage and absolute number of NK cells and IFN+Percentage ratio of NK cells Il1r8/Hepatocellular carcinoma tumor-bearing mice were high (fig. 3b, c and fig. 9c) finally, increased cytokine levels were observed in relation to a reduction in anti-tumor immunity (e.g. IFN) and the pro-inflammatory cytokines associated with tumor promotion (IL-6, tumor necrosis factor- α -1, CCL2, CXCL1) (table 1) most importantly, NK cell depletion eliminated IL1r8/Protection against hepatogenesis observed in mice (fig. 3b and fig. 9 d).
Evidence suggests that NK cells can inhibit hematopoietic metastasis5. In the spontaneous lung metastasis model of sarcoma (MN/MCA1), Il1r8/Mice showed a reduced number of blood cell metastases, while primary tumor growth was unaffected (fig. 3e and fig. 9e, f). In Il1r8/Total and mature CD27 in the lungIs low inNK cell frequency was higher (fig. 3 f). Lung metastasis at euthanasia and in vivo imaging analysis (FIGS. 3g and 9e) showed NK cell depleted Il1r8/Protection was completely abrogated in mice. In addition, IL-18 or IFN neutralization eliminates or significantly reduces targeting to Il1r8/Protection of the metastases observed in mice (fig. 9 g). In contrast, CD4+/CD8+Depletion of cells or IL-17A, or deficiency of IL-1R1 (involved in T helper 17 cell development) did not affect the phenotype (FIG. 9h, i).
Liver metastasis is a major problem in the progression of colorectal cancer. Therefore, the inventor uses the MC38 colon cancer strain21Evaluation of Il1r8/Protective capacity of NK cells against liver metastases. As shown in FIG. 3h, Il1r8/Mice were protected from liver metastases of MC38 colon cancer. In addition, IL-18 gene deficiency elimination Il1r8/Protection against liver metastases observed in mice (FIG. 9j), thus indicating that IL-1R 8-dependent control of MC 38-derived liver metastases occurred along the IL-18/IL-18R axis. To evaluate Il1r8/NK cells in cancer protectionThe main role in protection was the use of adoptive transfer (FIG. 9 k-m). Il1r8+/+Adoptive transfer of NK cells had no effect on lung and liver metastases. In contrast, Il1r8/Adoptive transfer of NK cells significantly and prominently reduced the number and volume of lung and liver metastases (fig. 3i, j and fig. 9 n). This observation is a possible translation hint based on the natural history of colon cancer and clinical challenges. Therefore, IL-1R8 gene inactivation relieves NK cell-mediated resistance to carcinogenesis in the liver and amplifies the anti-metastatic capacity of these cells in their spontaneous form of NK cells in the liver and lung.
Finally, the inventors investigated whether IL-1R8 affects the antiviral activity of NK cells, focusing on Mouse Cytomegalovirus (MCMV) infection22. As shown in FIG. 4a, the liver virus titer was Il1r8/Mouse middle ratio Il1r8+/+Low in mice, it was shown that IL-1R8 deficiency was associated with more efficient control of MCMV infection. IFN (Interferon IFN)+Frequency and degranulation of NK cells (i.e., CD107 a)+Frequency of NK cells) at Il1r8 1.5 days after infection/Significantly higher in spleen and liver of mice (fig. 4 b). 4.5 days after infection, IFN in spleen and liver due to better control of viral spread+And CD107a+NK cells were significantly reduced (fig. 4 b). Consistent with more efficient control of infection, at Il1r8/A decrease in the level of proinflammatory cytokines was observed in mice (figure 10 a). NK-cell adoptive transfer experiments in MCMV infected neonatal mice still not having mature NK cells12. As shown in FIG. 4c, Il1r8/Adoptive transfer of NK cells confers a ratio of Il1r8+/+Higher protection in NK cells, e.g. 4 out of 9 mice, had no detectable viral titers in the brain.
NK cells belong to the field of a complex diversity of natural lymphocytes (ILCs)23. Human and mouse non-NK ILCs express IL-1R8mRNA and protein (literature 24). Preliminary experiments were performed to assess the role of IL-1R 8in ILC function. Il1r 8in MCMV infection model/ILC1 shows increased IFN production but shows a smaller population compared to cells, and Il1r8/Of IFN-producing cells (fig. 4 d); they may therefore play an important role for the phenotype. These results provide IL-1R8 has initial evidence of a regulatory effect on ILC. Further studies are needed to assess their actual relevance to the ILC diverse population. Collectively, these results indicate that IL-1R8 deficient mice are protected against MCMV infection, protection being dependent on increased NK cell activation.
IL-1R8 deficiency is associated with exacerbation of inflammation and immune response under a variety of conditions1,10. NK cells are involved in the bidirectional interaction of macrophages, dendritic cells and lymphocytes3,4,25,26. Therefore, there is a need to examine the role of NK cells in inflammatory and autoimmune diseases associated with IL-1R8 deficiency1,10. IL-1R8 deficient mice show increased susceptibility to developing colitis and colitis-related azomethine oxide cancers27,28. The diverse effects of IL-1R8 deficiency in the small intestine and liver on carcinogenesis appear to reflect the fundamental, tissue-dominated differences in the immune mechanisms involved in carcinogenesis in these different anatomical sites. In particular, a large number of NK cells are present in the liver19This physiological characteristic of the organ appears to support this apparent diversity even more.
NK cells do not normally play a major role in the control of solid tumors6. In contrast, there is evidence that NK has a role in controlling blood lung metastases5,29. The results herein show that gene inactivation of IL-1R8 releases NK cells, resulting in suppression of liver carcinogenesis and protection against liver and lung metastases. IL-1R8 deficient mice display TLR exacerbation and IL-1 driven inflammation10Inflammation promoting carcinogenesis of the liver30. Therefore, our results may underestimate the ablation of the NK cell checkpoint IL-1R8 against hepatic primary and metastatic tumors. Thus, NK cells can restrict solid cancers and metastases, provided that a key validated checkpoint, such as IL-1R8, is removed, and tissue immunological profiles are taken into account.
Example 2
Materials and methods
In vitro functional activation of NK cells
Enrichment by negative magnetic separation (NK cell isolation kit II, gentle and gentle) (as described in example 1) Il1r8+/+And Il1r8-/-SpleenNK cells, cultured for 8 days in RPMI 10% FBS supplemented with IL-2(Peprotech,20ng/ml) plus IL-15(Peprotech,10ng/ml) (huangg BY et al, PloS ONE (2015). MC38 cells (as shown in example 1) were pre-treated with IFN γ (24 hours) to mimic the tumor microenvironment and induce PD-L1 expression as previously shown (Juneja VR et al, j.exp.med. (2017). NK cells were pre-treated with anti-PD-1 blocking antibody or related isotype control (both from BioxCell,1 μ g/ml) for 30 minutes (37 ℃). MC38 cells were washed and co-cultured with NK cells (1:2) for 3 hours. intracellular expression of IFN γ and granzyme B in NK cells was determined BY flow cytometry.
Results
In vitro Effect of IL-1R8 deficiency in combination with PD-1 blocking
The inventors herein show that upon contact with a tumor target expressing ligand (PD-L1), blockade of PD-1 drives increased NK cell activation in IL-1R8 deficient NK cells compared to wild-type NK cells, showing that the combination of IL-1R8 and PD-1 blockade contributes to NK cell effector function (fig. 12).
Example 3
Materials and methods
IL-1R8 expression in human T cells.
Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from peripheral blood of healthy donors by Ficoll density gradient centrifugation (GE medical biosciences) after approval by the ethical committee of the human manitas research hospital. IL-1R8 expression was determined by flow cytometry in T cell subsets according to the expression of CD3, CD4, CD8, CCR7, CD45RO, CD127, CD25 (Gattinoni l. et al, Nature Medicine (2011).
Proliferation assay
Negative magnetic separation (pan T cell separation kit II, gentle and gentle), enrichment of splenic T cells from Il1r8+/+ and Il1r 8-/-mice, and use of
Figure BDA0002550531970000351
CFDA SE dye (Invitrogen, 1 μ M) was preincubated for 10 minutes (37 ℃). T cells were washed in IMDM 10% FBS 0.1% BME (Gibco) plus Dynabeads mouse T-activator CD3/CD28 (Gibco, 1-bead X cells) plus IL-2 (Addison interleukin (Proleukin),20ng/ml), IL-12(Peprotech,20ng/ml), IL-18(MBL,20ng/ml)ng/ml) for two days (Hu b. et al, Cell Rep (2017); freeman B. et al, PNAS (2012)). CFDA SE and CD44 expression was determined by flow cytometry in CD 8T cells.
In vitro T cell activation
Il1r8+/+ and Il1r 8-/-murine spleen CD8+ T cells were enriched by negative magnetic separation (CD8a + isolation kit, mouse, gentle, Meitian) and cultured for 2 days in IMDM 10 % FBS 0, 1% BME (Gebucco) and Dynabeads mouse T-activator CD3/CD28 (Gebucco, 1 bead X cells) plus IL-2 (aldesleukin, 20ng/ml), IL-12(Peprotech,20ng/ml) alone or in combination. T cells were treated with IL-18(MBL,20ng/ml) (overnight) and stimulated with a cell stimulation mix (eBioscience) plus Golgi Plug (BD biosciences) as specified for 3 hours (Hu B. et al, CellRep (2017); Freeman B. et al, PNAS (2012)). intracellular expression of IFN γ and granzyme B in CD 8T cells was determined by flow cytometry.
Results
The inventors hypothesized that CD8+ T lymphocytes express IL-1R8, and function as a negative regulator in this cell type. The inventors first examined the expression of IL-1R 8in human T cells from healthy donors by flow cytometry. Here, the inventors showed that CD8+ T cells exhibited higher levels of IL-1R8 than CD4+ T cells. Furthermore, IL-1R8 expression was higher in the effector/memory T cell subset compared to naive T cells, showing that IL-1R8 expression is associated with the gain of effector capacity (FIG. 13). To illustrate the role of IL-1R 8in cytotoxic CD8+ T cells (with cytokines IL-2, IL-12 and IL-18), the inventors evaluated CD8+ T cell proliferation, maturation and in vivo activation following TCR stimulation, which is involved in CD8+ T cell activation. In fig. 14A, the inventors showed that Il1r8-/-CD8+ T cells showed a higher proliferation rate than CD8+ T cells of wild type mice. Consistent with this observation, the maturation marker CD44 for Il1R8-/-CD8+ T cells was upregulated compared to wild-type CD8+ T cells (fig. 14B), suggesting that Il-1R8 deficiency promotes CD8+ T cell expansion and conversion from naive to effector T cells. Finally the inventors showed that IFN γ and granzyme B production was enhanced in Il1R8-/-CD8+ T cells, whereas IL-1R 8-deficiency enhanced the response to IL-18 stimulation (FIGS. 15A-D). These results indicate that IL-1R8 gene silencing results in increased proliferation, maturation and activation of CD8+ T cells.
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Gentleman, R.C. et al, Bioconductor: open software development for computational and bioinformatics.
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42.
Figure BDA0002550531970000411
S.,
Figure BDA0002550531970000412
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Sequence listing
<110> Bo ai Mi La le Du Ltd (Humanitas miracole S.p.A.)
University of Mannich University (Humanitas University)
<120> NK or T cells and uses thereof
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uaccagagca gcacguugau u 21
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cuucccgucg uuuaucuccu u 21
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actttcgatg gtagtcgccg t 21
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cccaaggcca accgcgagaa gat 23
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gtcccggcca gccaggtcca g 21
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tggagtaaga ggacacttgc gaa 23
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<220>
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agtgtgccct gacctgaaag a 21
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<212>DNA
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tccaagagta tggcgccct 19
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ggctggcccg ataaggat 18
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gtccccagtc atcacagcg 19
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gaatgggact ttgggctttg 20
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gcttttcgtg gcagcagata c 21
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agagtttgat cctggctcag 20
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ggctgctggc acgtagttag 20
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Met Pro Gly Val Cys Asp Arg Ala Pro Asp Phe Leu Ser Pro Ser Glu
1 5 10 15
Asp Gln Val Leu Arg Pro Ala Leu Gly Ser Ser Val Ala Leu Asn Cys
20 25 30
Thr Ala Trp Val Val Ser Gly Pro His Cys Ser Leu Pro Ser Val Gln
35 40 45
Trp Leu Lys Asp Gly Leu Pro Leu Gly Ile Gly Gly His Tyr Ser Leu
50 55 60
His Glu Tyr Ser Trp Val Lys Ala Asn Leu Ser Glu Val Leu Val Ser
65 70 75 80
Ser Val Leu Gly Val Asn Val Thr Ser Thr Glu Val Tyr Gly Ala Phe
85 90 95
Thr Cys Ser Ile Gln Asn Ile Ser Phe Ser Ser Phe Thr Leu Gln Arg
100 105 110
Ala Gly Pro Thr Ser His Val Ala Ala Val Leu Ala Ser Leu Leu Val
115 120 125
Leu Leu Ala Leu Leu Leu Ala Ala Leu Leu Tyr Val Lys Cys Arg Leu
130 135 140
Asn Val Leu Leu Trp Tyr Gln Asp Ala Tyr Gly Glu Val Glu Ile Asn
145 150 155 160
Asp Gly Lys Leu Tyr Asp Ala Tyr Val Ser Tyr Ser Asp Cys Pro Glu
165 170 175
Asp Arg Lys Phe Val Asn Phe Ile Leu Lys Pro Gln Leu Glu Arg Arg
180185 190
Arg Gly Tyr Lys Leu Phe Leu Asp Asp Arg Asp Leu Leu Pro Arg Ala
195 200 205
Glu Pro Ser Ala Asp Leu Leu Val Asn Leu Ser Arg Cys Arg Arg Leu
210 215 220
Ile Val Val Leu Ser Asp Ala Phe Leu Ser Arg Ala Trp Cys Ser His
225 230 235 240
Ser Phe Arg Glu Gly Leu Cys Arg Leu Leu Glu Leu Thr Arg Arg Pro
245 250 255
Ile Phe Ile Thr Phe Glu Gly Gln Arg Arg Asp Pro Ala His Pro Ala
260 265 270
Leu Arg Leu Leu Arg Gln His Arg His Leu Val Thr Leu Leu Leu Trp
275 280 285
Arg Pro Gly Ser Val Thr Pro Ser Ser Asp Phe Trp Lys Glu Val Gln
290 295 300
Leu Ala Leu Pro Arg Lys Val Gln Tyr Arg Pro Val Glu Gly Asp Pro
305 310 315 320
Gln Thr Gln Leu Gln Asp Asp Lys Asp Pro Met Leu Ile Leu Arg Gly
325 330 335
Arg Val Pro Glu Gly Arg Ala Leu Asp Ser Glu Val Asp Pro Asp Pro
340345 350
Glu Gly Asp Leu Gly Val Arg Gly Pro Val Phe Gly Glu Pro Ser Ala
355 360 365
Pro Pro His Thr Ser Gly Val Ser Leu Gly Glu Ser Arg Ser Ser Glu
370 375 380
Val Asp Val Ser Asp Leu Gly Ser Arg Asn Tyr Ser Ala Arg Thr Asp
385 390 395 400
Phe Tyr Cys Leu Val Ser Lys Asp Asp Met
405 410
<210>30
<211>410
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<213> Intelligent (Homo sapiens)
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Met Pro Gly Val Cys Asp Arg Ala Pro Asp Phe Leu Ser Pro Ser Glu
1 5 10 15
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20 25 30
Thr Ala Trp Val Val Ser Gly Pro His Cys Ser Leu Pro Ser Val Gln
35 40 45
Trp Leu Lys Asp Gly Leu Pro Leu Gly Ile Gly Gly His Tyr Ser Leu
50 55 60
His Glu Tyr Ser Trp Val Lys Ala Asn Leu Ser Glu Val Leu Val Ser
65 70 75 80
Ser Val Leu Gly Val Asn Val Thr Ser Thr Glu Val Tyr Gly Ala Phe
85 90 95
Thr Cys Ser Ile Gln Asn Ile Ser Phe Ser Ser Phe Thr Leu Gln Arg
100 105 110
Ala Gly Pro Thr Ser His Val Ala Ala Val Leu Ala Ser Leu Leu Val
115 120 125
Leu Leu Ala Leu Leu Leu Ala Ala Leu Leu Tyr Val Lys Cys Arg Leu
130 135 140
Asn Val Leu Leu Trp Tyr Gln Asp Ala Tyr Gly Glu Val Glu Ile Asn
145 150 155 160
Asp Gly Lys Leu Tyr Asp Ala Tyr Val Ser Tyr Ser Asp Cys Pro Glu
165 170 175
Asp Arg Lys Phe Val Asn Phe Ile Leu Lys Pro Gln Leu Glu Arg Arg
180 185 190
Arg Gly Tyr Lys Leu Phe Leu Asp Asp Arg Asp Leu Leu Pro Arg Ala
195 200 205
Glu Pro Ser Ala Asp Leu Leu Val Asn Leu Ser Arg Cys Arg Arg Leu
210 215 220
Ile Val Val Leu Ser Asp Ala Phe Leu Ser Arg Ala Trp Cys Ser His
225230 235 240
Ser Phe Arg Glu Gly Leu Cys Arg Leu Leu Glu Leu Thr Arg Arg Pro
245 250 255
Ile Phe Ile Thr Phe Glu Gly Gln Arg Arg Asp Pro Ala His Pro Ala
260 265 270
Leu Arg Leu Leu Arg Gln His Arg His Leu Val Thr Leu Leu Leu Trp
275 280 285
Arg Pro Gly Ser Val Thr Pro Ser Ser Asp Phe Trp Lys Glu Val Gln
290 295 300
Leu Ala Leu Pro Arg Lys Val Gln Tyr Arg Pro Val Glu Gly Asp Pro
305 310 315 320
Gln Thr Gln Leu Gln Asp Asp Lys Asp Pro Met Leu Ile Leu Arg Gly
325 330 335
Arg Val Pro Glu Gly Arg Ala Leu Asp Ser Glu Val Asp Pro Asp Pro
340 345 350
Glu Gly Asp Leu Gly Val Arg Gly Pro Val Phe Gly Glu Pro Ser Ala
355 360 365
Pro Pro His Thr Ser Gly Val Ser Leu Gly Glu Ser Arg Ser Ser Glu
370 375 380
Val Asp Val Ser Asp Leu Gly Ser Arg Asn Tyr Ser Ala Arg Thr Asp
385390 395 400
Phe Tyr Cys Leu Val Ser Lys Asp Asp Met
405 410
<210>31
<211>410
<212>PRT
<213> Intelligent (Homo sapiens)
<400>31
Met Pro Gly Val Cys Asp Arg Ala Pro Asp Phe Leu Ser Pro Ser Glu
1 5 10 15
Asp Gln Val Leu Arg Pro Ala Leu Gly Ser Ser Val Ala Leu Asn Cys
20 25 30
Thr Ala Trp Val Val Ser Gly Pro His Cys Ser Leu Pro Ser Val Gln
35 40 45
Trp Leu Lys Asp Gly Leu Pro Leu Gly Ile Gly Gly His Tyr Ser Leu
50 55 60
His Glu Tyr Ser Trp Val Lys Ala Asn Leu Ser Glu Val Leu Val Ser
65 70 75 80
Ser Val Leu Gly Val Asn Val Thr Ser Thr Glu Val Tyr Gly Ala Phe
85 90 95
Thr Cys Ser Ile Gln Asn Ile Ser Phe Ser Ser Phe Thr Leu Gln Arg
100 105 110
Ala Gly Pro Thr Ser His Val Ala Ala Val Leu Ala Ser Leu LeuVal
115 120 125
Leu Leu Ala Leu Leu Leu Ala Ala Leu Leu Tyr Val Lys Cys Arg Leu
130 135 140
Asn Val Leu Leu Trp Tyr Gln Asp Ala Tyr Gly Glu Val Glu Ile Asn
145 150 155 160
Asp Gly Lys Leu Tyr Asp Ala Tyr Val Ser Tyr Ser Asp Cys Pro Glu
165 170 175
Asp Arg Lys Phe Val Asn Phe Ile Leu Lys Pro Gln Leu Glu Arg Arg
180 185 190
Arg Gly Tyr Lys Leu Phe Leu Asp Asp Arg Asp Leu Leu Pro Arg Ala
195 200 205
Glu Pro Ser Ala Asp Leu Leu Val Asn Leu Ser Arg Cys Arg Arg Leu
210 215 220
Ile Val Val Leu Ser Asp Ala Phe Leu Ser Arg Ala Trp Cys Ser His
225 230 235 240
Ser Phe Arg Glu Gly Leu Cys Arg Leu Leu Glu Leu Thr Arg Arg Pro
245 250 255
Ile Phe Ile Thr Phe Glu Gly Gln Arg Arg Asp Pro Ala His Pro Ala
260 265 270
Leu Arg Leu Leu Arg Gln His Arg His Leu Val Thr Leu Leu Leu Trp
275 280 285
Arg Pro Gly Ser Val Thr Pro Ser Ser Asp Phe Trp Lys Glu Val Gln
290 295 300
Leu Ala Leu Pro Arg Lys Val Gln Tyr Arg Pro Val Glu Gly Asp Pro
305 310 315 320
Gln Thr Gln Leu Gln Asp Asp Lys Asp Pro Met Leu Ile Leu Arg Gly
325 330 335
Arg Val Pro Glu Gly Arg Ala Leu Asp Ser Glu Val Asp Pro Asp Pro
340 345 350
Glu Gly Asp Leu Gly Val Arg Gly Pro Val Phe Gly Glu Pro Ser Ala
355 360 365
Pro Pro His Thr Ser Gly Val Ser Leu Gly Glu Ser Arg Ser Ser Glu
370 375 380
Val Asp Val Ser Asp Leu Gly Ser Arg Asn Tyr Ser Ala Arg Thr Asp
385 390 395 400
Phe Tyr Cys Leu Val Ser Lys Asp Asp Met
405 410

Claims (22)

1. An isolated human cell which is a natural killer cell (NK) or T cell, wherein said cell has a stable or transient defect in IL-1R8 expression and/or activity.
2. The cell of claim 2, wherein the T cell is a CD8+ T cell.
3. The cell of any one of claims 1-2, wherein the cell produces a greater amount of at least one anti-tumor immunity-related effector molecule than a cell expressing IL-1R 8.
4. The cell of claim 3, wherein the molecule is interferon-gamma (IFN- γ) and/or granzyme B and/or FasL.
5. The cell of any one of claims 1-4, further being deficient in the expression and/or activity of at least one checkpoint of NK cell maturation and/or effector function.
6. The cell of claim 5, wherein said at least one checkpoint of NK cell maturation and/or effector function is selected from the group consisting of: CIS, KIRs, PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT.
7. A cell population comprising NK cells and/or T cells according to any one of claims 1 to 6.
8. A composition comprising a cell according to any one of claims 1 to 6 or a population of cells according to claim 7, preferably further comprising at least one physiologically acceptable carrier.
9. The cell according to any one of claims 1 to 6, or the population according to claim 7, or the composition according to claim 8, for use as a medicament, preferably for the treatment and/or prevention of tumors and/or metastases, or microbial or viral infections.
10. The cell or population or composition for use according to claim 9, for Adoptive Cell Transfer (ACT), cell therapy, mismatched bone marrow transplantation, mismatched NK cell infusion or cytokine-induced killing (CIK) cell infusion.
A repressor or inhibitor of IL-1R8 expression and/or activity for use in medicine.
12. A repressor or inhibitor of IL-1R8 expression and/or activity for use in the treatment and/or prevention of tumours and/or metastases, or microbial or viral infection.
13. The repressor or inhibitor for use according to claim 11 or 12, wherein the repressor or inhibitor is at least one molecule selected from the group consisting of:
a) an antibody or fragment thereof;
b) a polypeptide;
c) a small molecule;
d) a polynucleotide encoding the antibody or polypeptide or functional derivative thereof;
e) polynucleotides, such as antisense constructs, antisense oligonucleotides, RNA interference constructs or siRNA,
e) a vector comprising or expressing a polynucleotide as defined in d) or e);
f) CRISPR/Cas9 components, such as sgrnas;
g) a host cell genetically engineered to express said polynucleotide or antibody, or which comprises at least one of the components of the polynucleotide defined in d) or e) or f),
preferably the polynucleotide is an RNA inhibitor, preferably selected from: siRNA, miRNA, shRNA, stRNA, snRNA, and antisense nucleic acids, more preferably the polynucleotide is at least one siRNA selected from AGU UUC GCG AGC CGA GAU CUU (SEQ ID NO: 1); UAC CAG AGC AGC ACG UUG AUU (SEQ ID NO: 2); UGA CCC AGG AGU ACU CGU GUU (SEQ ID NO: 3); CUU CCC GUC GUU UAU CUC CUU (SEQ ID NO:4) (all 5.
14. A repressor or inhibitor for use according to any one of claims 11 to 13 for use in NK and/or T cells.
15. The repressor or inhibitor for use according to any one of claims 11 to 14, for use in Adoptive Cell Transfer (ACT), cell therapy, mismatched bone marrow transplantation, mismatched NK cell infusion or cytokine-induced killer (CIK) cell infusion.
16. A pharmaceutical composition comprising a repressor or inhibitor according to any of claims 11 to 13 and at least one pharmaceutically acceptable carrier, and optionally a therapeutic agent.
17. A cell or population or composition for use according to any one of claims 9 to 10, or a repressor or inhibitor for use according to any one of claims 12 to 15, wherein:
a) the tumor is preferably a solid tumor or a hematological tumor, preferably selected from the following: colon/rectal cancer, adrenal cancer, anal cancer, biliary tract cancer, bladder cancer, bone cancer, adult brain/CNS tumor, childhood brain/CNS tumor, breast cancer, male breast cancer, unknown primary cancer, castleman's disease, cervical cancer, endometrial cancer, esophageal cancer, ewing's tumor family, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, hodgkin's disease, kaposi's sarcoma, kidney cancer, larynx and hypopharyngeal cancer, leukemia, Acute Lymphocytic Leukemia (ALL), acute myelogenous leukemia (AML, including myelogenous sarcoma and skin leukemia), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous (CML) leukemia, chronic myelomonocytic leukemia (ml), childhood leukemia, liver cancer, lung cancer, non-small cell lung cancer, lung carcinoid tumor, lymphoma, skin lymphoma, cutaneous lymphoma, small cell lung cancer, colon cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, adenoid cystic carcinoma of the nasal cavity and sinuses, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma in children, oral and oropharyngeal carcinoma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, penile carcinoma, pituitary tumor, prostate carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma-adult soft tissue carcinoma, skin carcinoma-basal and squamous cells, skin carcinoma-melanoma, skin carcinoma-Merkel cells, small intestine carcinoma, stomach carcinoma, testicular carcinoma, thymus carcinoma, thyroid carcinoma, uterine sarcoma, uveal melanoma, vaginal carcinoma, vulval carcinoma, Waldenstrom's macroglobulinemia, Wilms ' tumor, more preferably solid tumor, preferably colorectal cancer, metastasis is lung or liver metastasis, or
b) Infection is caused by one of the following viruses or bacteria: herpes viruses, preferably cytomegalovirus, Human Immunodeficiency Virus (HIV), Hepatitis C Virus (HCV), Hepatitis B Virus (HBV), West Nile Virus (WNV), Salmonella, Shigella, Legionella, mycobacteria.
18. A method of obtaining the cell of any one of claims 1-6, or the population of claim 7, or the composition of claim 8, comprising stably or transiently inhibiting expression and/or function of IL-1R 8in a population of NK or T cells or cells comprising NK and/or T cells, and optionally expanding the silenced population in vitro.
19. The method of claim 18, wherein the T cell is a CD8+ T cell.
20. The method of claim 18 or 19, wherein the NK or T cell or cell population is preferably previously purified from isolated Peripheral Blood Mononuclear Cells (PBMCs), and optionally expanded in vitro preferably with rhIL-2.
21. The method of claim 18 or 19 or 20, further comprising inhibiting or suppressing expression and/or function of at least one other checkpoint of NK cell maturation and/or effector function.
22. The method of claim 21, wherein the at least one checkpoint of NK cell maturation and/or effector function is selected from the group consisting of: CIS, KIRs, PD-1, CTLA-4, TIM-3, NKG2A, CD96, TIGIT.
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